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University Microfilms International 300 N. Zeeb Road Ann Arbor, Ml 48106 8315482 MacGuidwin, Ann Elizabeth PATHOGENICITY AND ECOLOGY OF MELOIDOGYNE HAPLA ASSOCIATED WITH ALLIUM CEPA Ph.D. 1983 Michigan State University University Microfilms International 300 N. Zeeb Road, Ann Arbor, MI 48106 PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V 1. Glossy photographs or pages______ 2. Colored illustrations, paper or print_____ 3. Photographs with dark background_____ 4. Illustrations are poor copy______ 5. Pages with black marks, not original copy______ 6. Print shows through as there is text on both sides of page______ 7. Indistinct, broken or small print on several pages 8. Print exceeds margin requirements_____ 9. Tightly bound copy with print lost in spine______ 1 0. Computer printout pages with indistinct print . 11 . Page(s)__________ lacking when material received, and not available from school or author. 12 . Page(s)___________ :seem to be missing in numbering only as text follows. 13. Two pages numbered I__________ . Text follows. 14. Curling and wrinkled pages 15. Other University Microfilms International PATHOGENICITY AND ECOLOGY OF Meloidogyne hapla ASSOCIATED WITH Allium cepa by Ann Elizabeth MacGuidwin A DISSERTATION Subm itted to Michigan S ta te University in partial fulfillm ent of the requirem ents for the degree of DOCTOR OF PHILOSOPHY D epartm ent of Entomology 1983 ABSTRACT PATHOGENICITY AND ECOLOGY OF Meloidogyne hapla ASSOCIATED WITH Allium cepa by Ann Elizabeth MacGuidwin Meloidogyne hapla Chitwood was the third m ost prevalent phytoparasitic nem atode observed in Michigan onion acreage surveyed in 1980. A positive linear relationship betw een M. hapla density and th e final yield of Allium cepa L. cv Krummery Special was observed in field trials. This nem atode also inhibited the growth of A. cepa under greenhouse conditions when plants w ere exposed to 15,000 or more M. hapla eggs a t seeding. Another cultivar, Downing Yellow Globe, was more tolerant of M. hapla parasitism . M. hapla reproduced on A. cepa in greenhouse and field environm ents. One com plete generation of M. hapla per year was observed on A. cepa and nem atode populations increased from the beginning to end of a growing season. The increase in M. hapla population levels was less for nem atodes associated with A. cepa than for Daucus caro ta L., due primarily to the low number of nematodes invading A. cepa root tissue rath er than to decreased survival or reproduction. Spores of vesicular-arbuscular mycorrhizal fungi were present in all sites surveyed in 1980. Several experim ents were conducted to determ ine th e life stage of M. hapla most affected by the presence of the mycorrhizal fungus, Glomus fasciculatus (Thaxter) Gerd. & Trappe. The penetration of second-stage M. hapla into roots was decreased in A. cepa colonized by G. fasciculatus. Mycorrhizal colonization slightly impeded the development of M. hapla juveniles within roots, but did not influence the number of eggs produced per adult fem ale. In the greenhouse te sts, th e growth of A. cepa colonized by mycorrhizae and nem atodes was not different from th a t of plants inhabited by nem atodes alone. Conversely, mycorrhizal A. cepa transplanted to microplots in the field were more tolerant and supported higher nematode densities than nonmycorrhizal plants. The discrepant results betw een similar experim ents conducted in the greenhouse and field were probably due to the methodology employed, but also re fle c t the complexity of the relationship between nem atodes and plant growth, and the need for continued research on the interaction between nem atodes and other components of agroecosystem s. ACKNOWLEDGMENTS I would like to thank my major professor, Dr. George Bird, for his excellent guidance and support during my graduate studies a t Michigan S ta te University. My appreciation is also extended to the members of my com m ittee, Drs. Gene Safir, Dean Haynes, and Stuart Gage, for th eir tim e, assistance, and enthusiasm in sharing their knowledge and experiences with m e. I have enjoyed and benefited from my association with the faculty, staff, and students of th e Entomology D epartm ent. In particular, I would like to acknowledge my respect and gratitude to Dr. Jam es Bath for his outstanding leadership and ability to instill a sense of professionalism and cam araderie in all members of this departm ent. I also thank Mr. Kenneth Dimoff, Ms. Susan B attenfield, Mr. Thomas Ellis, and countless other staff members for their patience and assistance during my graduate program. All members of the "Nematology Group" deserve special thanks for their friendship and support of my research e ffo rts. Mr. John Davenport was especially instrum ental in making my stay a t MSU both enjoyable and fruitful. Mr. Fred Warner and Mr. Rhet F rem et were two of the many able and enthusiastic employees who assisted me in my laboratory and field experim ents. My sincere thanks are also extended to Ms. Barbara Mullen for her outstanding contribution to all phases of my research and in particular for her e ffo rts in the preparation of this m anuscript. Finally, I would like to convey my appreciation and love for my husband, Reid, and my parents, Frances and Bill Latoza. Their endless encouragem ent, understanding, and support contributed immeasurably to the fulfillm ent of my educational goals. TABLE OF CONTENTS I. In tro d u c tio n ......................................................................................... *............... 1 II. Objectives ............. III. Rationale and Research Approach ..................................................................... 3 IV. Background............................................................................................................ 10 2 A. Life H is to ry .................................................................................................10 B. Pathogenicity of Meloidogyne h a p ia .......................................................1 C. Interaction Between Meloidogyne hapla and Abiotic Factors ...................................................................................16 1. Tem perature ........................................................................................16 2. M o istu re ................................................................................................ 19 D. 3. Soil T e x tu r e ......................................................................................... 21 k. Soil pH .................................................................................................. 21 Interaction Between Meloidogyne hapla and Biotic F a c to rs .......................................................................................22 1. Predators and P a r a s ite s .................................................................... 22 E. 2. Other Phytoparasitic N e m a to d e s ................................................... 23 3. Vesicular-Arbuscular M y co rrh iz ae ................................................. 25 A Computer Model Simulating the Population Dynamics of Meloidogynehapla Associated with Allium Cepa ........................... 28 1. Overview of the M o d el...................................................................... 29 2. Description of Subroutines................................................................ 29 a. Subroutine O n io n ........................................................................ 29 b. Subroutine N e m fe e d ..................................................................33 c. Subroutine N e m a ........................................................................ 33 d. Subroutine S o ilte m p ................................................................. 37 ill TABLE OF CONTENTS, continued e. 3. Subroutine Model 1, Model 2, Model 3 ................................. 37 Model O u tp u t....................................................................................... 39 V. Problem Form ulation........................................................................................... 39 VI. System Identification and Mensuration of System C o m p o n en ts................ 40 VII. E x p e rim e n ta tio n ..................................................................................................48 C hapter I. Studies on the O ccurrence, Pathogenicity, and Increase of Meloidogyne hapla associated with Allium c e p a .................... 48 A. B. C. Survey of Michigan Onion A creage for Meloidogyne hapla and O ther Phytoparasitic Nem atodes ....................................... 49 1. In tro d u ctio n .........................................................................................49 2. M aterials and M eth o d s......................................................................49 3. R e s u lts ..................................................................................................52 4. D iscussion............................................................................................. 65 Pathogenicity and Increase of Meloidogyne hapla Infecting Allium cepa as Influenced by Initial Inoculum L e v e l .......................................................................................... 69 1. In tro d u ctio n ......................................................................................... 69 2. M aterials and M eth o d s......................................................................70 3. R e s u lts .......................... 4. Discussion..............................................................................................97 73 Pathogenicity and Increase of Meloidogyne hapla on Two Onion C ultivars, Two C arro t C ultivars, and S u d a x ................ 102 1. Introduction .................... 102 2. M aterials and M ethods.................................................................... 103 3. R e s u lts ................................................................................................ 104 4. Discussion............................................................................................120 TABLE OF CONTENTS, continued D. E. Stage-specific Survival of Meloidogyne hapla A ssociated With Allium c e p a ................................................................. 124 1. In tro d u c tio n ....................................................................................... 124 2. M aterials and M e th o d s.................................................................... 125 3. R esults.................................................................................................. 127 4. D iscussion............................................................................................ 132 General Discussion ................................................................................. 133 C hapter n . Studies on the Interaction Between Meloidogyne hapla and th e Vesicular-Arbuscular M ycorrhizal Fungus, Glomus fasciculatus, Associated with Allium c e p a .......................... A. B. C. 136 Influence of the Vesicular-Arbuscular Mycorrhizal Fungus, Glomus fasciculatus, on th e Pathogenicity and Increase of Meloidogyne hapla Infecting Allium c e p a .......................................... 137 1. Introduction.......................................................................................... 137 2. M aterials and M eth o d s.................................................................... 138 3. R e s u lts .................................................................................................141 4. Discussion ......................................................................................... 154 Influence of Glomus fasciculatus on the Location and Penetration of Allium cepa roots by Meloidogyne h a p la ................ 161 1. In tro d u ctio n ........................................................................................ 161 2. M aterials and M eth o d s.................................................................... 161 3. R e s u lts .................................................................................................164 4. Discussion............................................................................................ 169 Influence of Glomus fasciculatus on the Stage-specific Development and Reproduction of Meloidogyne hapla Inhabiting Roots of Allium c e p a ........................................................... 174 v TABLE OF CONTENTS, continued D. 1. Introduction....................................................................................... 174 2. M aterials and M eth o d s.................................................................. ) 7 5 3. R e s u lts ............................................................................................. 1 7 8 tt. D iscussion......................................................................................... 190 General D iscussio n ................................................................................ 194 VIII. Summary and C onclusions............................................................................ 197 IX. Appendix Is Computer M o d el......................................................................201 X. Appendix II: Efficiency of a Method for Extracting Meloidogyne hapla from S o i l................................................... 223 XI. L iterature C i t e d ............................................................................................. 227 vf LIST OF TABLES 1 2 3 4 5 6 7 8 Incidence of phytoparasitic nem atodes in Michigan onion acreage surveyed in 1980................................................................ .54 Incidence of six nem atode genera in Michigan onion acreage in May, July, and Septem ber of 1980........................................... ,55 Incidence of six nem atode genera in five Michigan counties in Septem ber, 1980........................................................................... ,56 Sim ilarity in the incidence of phytoparasitic nem atode genera present in five Michigan counties in Septem ber, 1980 according to Sorensen's (A) and Bray and C urtis' (B) Index of Sim ilarity............................................................................................ 58 Incidence of Meloidogyne hapla in Michigan onion acreage surveyed in 1980............................................................................... 60 Allium cepa cultivar planted in sites infested with Meloidogyne hapla............................................................................ 61 Crop planted in 1979 in sites infested with Meloidogyne hapla in 1980...................................................................................... 62 N utrient analysis for soil samples containing Meloidogyne 63 9 10 11 12 13 C orrelation m atrix between Meloidogyne hapla density in September and soil nutrient level, two phytoparasitic nematode genera and vesicular-arbuscular mycorrhizal spores.................................................................................................. 64 Sampling schedule for Experim ent 2: Im pact of high initial inoculum levels of Meloidogyne hapla on Allium cepa............ 72 Root weight and a re a of two onion cultivars inoculated with one to five levels of Meloidogyne hapla or noninoculated.... 78 Fresh and dry weights and areas of leaves of two onion cultivars inoculated with one of five levels of Meloidogyne hapla or noninoculated.................................................................... 83 Bulb weight and volume of two onion cultivars inoculated with one of five levels of Meloidogyne hapla or noninoculated.................................................................................... 87 vl i LIST OF TABLES, continued Ik C orrelation m atrix between final density of Meloidogyne hapla per Allium cepa root system and nem atode density and length of root system s throughout the experim ent...................................... 9** 15 Number of Meloidogyne hapla detected in 100 cm soilfrom five crops on five sampling d ates....................................................................105 16 Number of Meloidogyne hapla juveniles and adults in 0.25 g of roots from five crops on th ree sampling d ates....................................... 106 17 C oefficients for the multiple linear regression of final Meloidogyne hapla density associated with Daucus caro ta and six biotic variables......................................................................................108 18 C oefficients for the multiple linear regression of final Meloidogyne hapla density associated with Allium cepa and six biotic v a ria b le s. ................................................................................109 19 C orrelation m atrix for final Meloidogyne hapla density associated with Daucus caro ta and six biotic variables............................ 110 20 C orrelation m atrix for final Meloidogyne hapla density associated with Allium cepa and six biotic variables................................ I l l 3 Number of Pratylenchus spp. d etected in 100 cm soil from five crops on five sampling d ates......................................................... .. 112 3 Number of Paratylenchus spp. detected in 100 cm soil from five crops on five sampling d ates................................................................... Ill* 21 22 3 23 Number of predaceous nem atodes (F. Mononchidae) in 100 cm soil samples from five crops on five sampling d ates........................ 115 2k N um ber of fungivorous nem atodes (SF. Aphelenchoidea) in 100 cm soil from five crops on five sampling d ates.................................116 25 Number of VAM spores (Glomus and Gigaspora spp.) in 100 cm soil from five crops on five sampling d ates........................................ 117 26 Regression statistic s for the relationship between Meloidogyne and/or Pratylenchus spp. and the weight of Allium cepa......................................................................................................... 119 27 Number and fresh weight (kg) of two carro t cultivars per 6.15 m eter row................................................................................................... 121 28 Incidence of Meloidogyne hapla in 1981 by life-stag e............................. 129 viit LIST OF TABLES, continued 29 Density of second-stage Meloidogyne hapla (log. q) in Allium cepa inoculated with nem atodes alone or with nem atodes and Glomus fasciculatus six and eight weeks following inoculation........................................................................................................ 146 30 Analysis of variance for Meloidogyne hapla density within Allium cepa tre a ted with Glomus fasciculatus, phosphorus, or tap w ater................. 166 31 Summary of the differences in the number of Meloidogyne hapla observed in soil samples from Allium cepa inoculated w ith Glomus fasciculatus. amended with phosphorus, or noninoculated and unamended....................................................................... 168 32 Root and shoot fresh weights (g) of Allium cepa inoculated with Glomus fasciculatus. amended with phosphorus, and noninoculated and unamended control......................................................... 170 33 Degree hours (base 9 C) accum ulated a t the first appearance of five life stages of Meloidogyne hapla associated with Allium cepa. ..................................................................................................... 182 34 Degree hour (base 9 C) requirem ents for the development of the median individual or 50% of th e population of Meloidogyne hapla associated with Allium cepa to a tta in five life stages.................................................................................................. 187 ix LIST OF FIGURES 1 C onceptualization of the M. hapla - A. cepa ecosystem with M. hapla as the reference point......................................................................... 11 2 Overview of a com puter model simulating th e population dynamics of Meloidogyne hapla associated with Allium cep a....................30 3 Flow chart of a com puter model sim ulating the population dynamics of Meloidogyne hapla associated with Allium cep a....................31 k Flow chart of Subroutine Onion........................................................................ 32 5 Flow chart of Subroutine Nema from the egg stage until the penetration of Allium cepa roots by Meloidogyne hapla juveniles..................................................................................................................35 6 Flow chart of Subroutine Nema: the root-inhabiting stages of Meloidogyne hapla. .........................................................................................36 7 Flow chart of Subroutine Nema: adult stages of Meloidogyne hapla. .............................................................................................38 8 Relationship between fresh and dry leaf weights of Krummery Special (A) and Downing Yellow Globe (B) Allium cepa grown in the greenhouse............................................................................43 9 Relationship between fresh and dry leaf weights of Krummery Special Allium cepa grown under field conditions................... 44 10 Location and number of sites in five counties in Michigan surveyed for phytoparasitic nem atodes in 1980............................................ 50 11 Relationship between inoculum density of Meloidogynehapla eggs and fresh weight of Downing Yellow Globe (A) and Krummery Special (B) Allium cepa. ............................................................... 74 12 Relationship between th e number of Meloidogyne hapla recovered per 1.0 gram root tissue and fresh weight of Downing Yellow Globe (A) and Krummery Special (B) Allium cepa......................................................................................................................... 75 13 Relationship between fresh weight of Krummery Special Allium cepa and inoculum density of Meloidogyne hapla including (A) and excluding (B) an inoculum level of 30,000 eggs per plant........................................................................................................ 76 x LIST OF FIGURES, continued 14 Fresh root weight of Krummery Special Allium cepa inoculated with one of five levels of Meloidogyne hapla or noninoculated......................................................................................................... 79 15 Root length (cm) of Krummery Special Allium cepa inoculated with one of five levels of Meloidogyne hapla or noninoculated.........................................................................................................81 16 Fresh shoot weight of Krummery Special Allium cepa inoculated with one of five levels of Meloidogyne hapla or noninoculated........................................................................................................ 85 17 Levels of Meloidogyne hapla eggs and juveniles in 100 cm of soil surrounding Allium cepa inoculated with one of five levels of nem atodes or noninoculated..............................................................88 18 Levels of Meloidogyne hapla juveniles in 100 cm of soil 4604 degree hours a fte r inoculation and planting of Allium cepa......................................................................................................................... 89 19 Relationship between the inoculum density of Meloidogyne hapla eggs and the number of nem atodes recovered per gram root tissue on Downing Yellow Globe (A) and Krummery Special (B) Allium cepa.......................................................... ........................... 91 20 Number of Meloidogyne hapla in 0.1 gram of Allium cepa roots inoculated with one of five levels of nem atodes or noninoculated........................................................................................................ 92 21 Levels of Meloidogyne hapla juveniles and adults in Allium cepa inoculated with one of five levels of nem atodes or noninoculated........................................................................................................ 93 22 Relationship between egg production by Meloidogyne hapla fem ales and to ta l nem atode density per Allium cepa root system .................................................................................................................... 96 23 Param eters descriptive of the relationship between plant growth and preplant nem atode density. Tolerance threshold (T), minimum yield (m)....................................................................................... 99 24 Number and fresh weight of Krummery Special (KS) and Downing Yellow Globe (DYG) Allium cepa produced per 6.15 m row....................................................................................................................118 25 Levels of Meloidogyne hapla in 100 cm^ soil samples collected from the M.S.U. Muck Research Farm in 1981.........................128 3 3 LIST OF FIGURES, continued 26 Stage-specific density of .Meloidogyne hapla inhabiting roots of Allium cepa in 100 cm of soil from th e M. S. U. Muck Research Farm in 19S1. *.................................................................................. 130 27 Relationship between Meloidogyne hapla density and the fresh weight of bulbs of Allium cepa harvested per 30 cm row..........................................................................................................................131 28 Mean fresh weight (+ standard error) of Allium cepa inoculated with Meloidogyne hapla, Glomus fasciculatus, Meloidogyne hapla and Glomus fasciculatus. and noninoculated control.........................................................................................142 29 Mean number of Meloidogyne hapla (+ standard error) inhabitating root systems of Allium cepa inoculated with Meloidogyne hapla alone (A) or with Meloidogyne hapla and Glomus fasciculatus (B)...................................................... ............................. 144 30 Proportion of the to ta l number of Meloidogyne hapla reaching the adult stage in Allium cepa inoculated with Meloidogyne hapla alone or with Meloidogyne hapla and Glomus fasciculatus (mean values + C l g o 5^*............................................ 147 31 Number of Meloidogyne hapla second-stage juveniles in 100 cm soil surrounding Allium cepa inoculated with Meloidogyne hapla alone or with Meloidogyne hapla and Glomus fasciculatus.......................................................................................... 148 32 Mean number (+ standard error) of Glomus fasciculatus in 100 cm soil surrounding Allium cepa inoculated with Meloidogyne hapla alone or with Meloidogyne hapla and Glomus fasciculatus. ........................................................................................ 149 33 Fresh bulb weight of Allium cepa supplemented with phosphorus, inoculated with Glomus fasciculatus (myco), amended with both Glomus fasciculatus and phosphorus, and unamended control, with and without th e nem atode Meloidogyne hapla. .............................................................................................150 34 Fresh shoot weight of Allium cepa supplemented with phosphorus, inoculated with Glomus fasciculatus (myco), amended with both Glomus fasciculatus and phosphorus, and unamended control, with and without the nem atode Meloidogyne hapla. .............................................................................................152 35 Number of Meloidogyne hapla in 0.3 gram of roots of Allium cepa supplemented with phosphorus, inoculated with Glomus fasciculatus. or amended with both Glomus fasciculatus and phosphorus, and unamended control............................................................... 155 x ii LIST OF FIGURES, continued 36 3 Number of Meloidogyne hapla juveniles in 100 cm of soil surrounding Allium cepa supplemented with phosphorus, inoculated with Glomus fasciculatus, amended with both Glomus fasciculatus and phosphorus, and unamended control................. 156 37 Number of Glomus fasciculatus spores in 100 cm of soil surrounding Allium cepa supplemented with phosphorus, inoculated with Glomus fasciculatus (mycorrhizae), amended with both Glomus fasciculatus and phosphorus, and unamended control, with and without th e nematode Meloidogyne hapla............................................................................................. 157 38 Number of Meloidogyne hapla within root system s of Allium cepa supplemented with phosphorus (phosphorus control^ inoculated with Glomus fasciculatus (mycorrhizae), or unamended (w ater control).................. 165 39 Number of Meloidogyne hapla per cen tim eter, 0.1 gram , or root tip in the root systems of Allium cepa supplemented with phosphorus (phosphorus control), inoculated with Glomus fasciculatus (mycorrhizae), and unamended control....................167 40 Stage-classes (1,2....... 6) used to classify Meloidogyne hapla inhabiting Allium cepa roots............................................................................ 177 41 Incidence of five stage-classes of Meloidogyne hapla inhabiting Allium cepa supplemented with phosphorus.............................. 179 42 Incidence of five stage-classes of Meloidogyne hapla inhabiting Aliium cepa inoculated with Glomus fasciculatus. ................. 180 43 Incidence of five stage-classes of Meloidogyne hapla inhabiting Allium cepa unamended with phosphorus and noninoculated with Glomus fa s c ic u la tu s.................................................... 181 44 Stage-specific incidence of the median individual (or 30% of the population) of Meloidogyne hapla inhabiting Allium cepa unamended with phosphorus or Glomus fasciculatus (control)*..............................................................................................................184 45 Stage-specific incidence of the median individual (or 50% of the population) of Meloidogyne hapla inhabiting Allium cepa supplem ented with phosphorus ..........................................................185 46 Stage-specific incidence of the median individual (or 50% of the population) of Meloidogyne hapla inhabiting Allium cepa inoculated with Glomus fasciculatus............................................................. 186 xi 11 LIST OF FIGURES, continued 47 Number of eggs produced per ovipositing Meloidogyne hapla fem ale inhabiting Allium cepa supplemented with phosphorus, inoculated with Glomus fasciculatus (mycorrhizae), and unamended control.......................................................... 188 48 Fresh weight of the roots of Allium cepa supplemented with phosphorus, inoculated with Glomus fasciculatus (mycorrhizae), and unamended control.......................................................... 189 49 Number of Meloidogyne hapla in the root system s of Allium cepa supplemented with phosphorus, inoculated with Glomus fasciculatus (mycorrhizae), and unamended control...................................191 50 Number (A) and percentage (B) of Meloidogyne hapla recovered per 100 cm soil given initial densities of 1 - 200 nem atodes............................................................................................................225 x tv INTRODUCTION Nematodes in the genus Meloidogyne are among the most economically im portant pathogens of food crops (Taylor and Sasser, 1978). C ollectively known as root-knot nem atodes, 37 species of this genus were recognized as of 1978 (Taylor and Sasser, 1978). All Meloidogyne spp. are phytoparasites and a lte r th e physiology and ontogeny of host plants. Reports of reduced plant growth, yield, and m arketability due to parasitism by Meloidogyne spp. are abundant in nem atological literatu re. In addition to a high level of pathogenicity, the global distribution and wide host range of Meloidogyne spp. contribute to the pest statu s of this nem atode. Plant stress and resulting crop loss caused by nem atodes may be excessive in some years and minimal in others due, in p a rt, to th e dynamics of nem atode populations. Plant growth in the presence of Meloidogyne hapla and other phytoparasitic nem atodes is related to nem atode density (Barker and O lthof, 1976). The abundance of ftf. hapla. in turn, influences th e popultion dynamics and pathogenicity of many nem atode species and other organisms contributing to plant damage. For example, parasitism of plants by M. hapla may increase the incidence of fungal and b acterial diseases (Griffin e t al., 1968; Jenkins and Coursen, 1957; Meagher and Jenkins, 1970). The population dynamics of M. hapla is influenced by a number of environm ental factors and complex biological associations. The abundance and distribution of this nematode are related to soil type, inter-specific com petition, soil biota, clim atic conditions, and m anagem ent practices (Norton, 1978; Wal­ lace, 1973). To adequately assess the population dynamics of M. hapla, it is necessary to understand the nature of th e association betw een these facto rs and the life history of this nem atode. 1 2 Study of the ecology of nem atodes was placed in highest priority by the ISCPP-ESCOP IPM R esearch Priority R eport (1979) and will contribute to th e developm ent of economically and biologically sound nem atode management programs. An understanding of events contributing to fluctuations in nem atode population levels will enhance models predicting crop loss due to nematode parasitism . Knowledge concerning the association betw een nem atode density and other biotic and abiotic components of an agroecosystem can form the basis for m anagem ent paradigms; th a t is, th e deletion, addition, or alteratio n of components within the agroecosystem . The purpose of this research was to examine th e growth and pathogencity of M. hapla populations associated with Allium cepa L., with particular emphasis on population phenomena occurring within a single nem atode generation. The influence of one biotic component of th e onion agroecosystem , vesiculararbuscular m ycorrhizae (VAM), on the ontogeny of M. hapla infecting a single A. cepa plant was assessed. Studies on th e nematode-VAM association w ere designed to provide the basis for further investigations on the dynamics of M. hapla populations in Michigan. This research was conducted in cooperation with entom ologists and plant pathologists who examined other biological components of the onion agroecosystem . OBJECTIVES The major goals of this research w ere (I) to elucidate and quantify the w ithin-generation dynamics of M. hapla and (2) to assess th e influence M. hapla in an onion agroecosystem both alone and in the presence of the VAM, Glomus fasciculatus. Specifically, th e objectives of this research were to: 3 1. examine the occurrence and pathogenicity of M. hapla associated with A. cepa in Michigan and th e suitability of this host for the increase of M. hapla populations 2. describe the within-generation dynamics of M. hapla infecting A. cepa 3. assess the influence of vesicular-arbuscular mycorrhizal fungi on the ontogeny and pathogenicity of M. hapla 4. re la te the findings of this research to data from previous studies in a com puter simulation model RATIONALE AND RESEARCH APPROACH Numerous studies have measured changes in nem atode population density, but few have elucidated th e forces responsible fo r fluctuating population levels. The density of nem atode populations has most often been measured to evaluate th e efficacy of various m anagem ent ta c tic s such as pesticide application, crop rotation, and tillage regim es. The inadequacy of many of the experim ents purporting to address the population dynamics of nem atodes is due to the exclusion of factors other than nem atodes in the experim ental design, and the use of sampling schemes inadequate for characterizing the spatial and tem poral distribution of nem atodes within a growing season. Consequently, it is not often dem onstrated if th e m anagem ent ta c tic kills nem atodes directly, or indirectly by altering some other component of the environm ent, or if the ta c tic influences the en tire nem atode population, or if only certain age classes are affected . Inform ation on the biology and ontogeny of nem atodes is a necessary supplement to th e study of nem atode population dynamics. Although com puter models simulating the life history of Meloidogyne arenaria (Ferris, 1976), 4 Pratvlenchus hexincisus (McSorley,1977), and H eterodera schachtii (Caswell e t al., 1981) have been developed, th ere is still an inadequate understanding among nem atologists for population phenomena occurring within a single nematode generation; developm ental rates, m ortality ra te s, reproductive ra te s, over­ w inter survival, e tc . The within-generation population dynamics of nematodes is of in terest for several reasons. D ata on th e stage-specific survival of nem atodes can be used to estim ate the optimum dates of pesticide application, harvest, e tc . Information on the age structure of a nem atode population throughout th e growing season can guide sampling activities for more accu rate population m easurem ent. Prediction of end-of-season nem atode densities can influence m anagem ent decisions for th e subsequent growing season, such as crop selection or fum igation. The M. hapla-A. cepa system was used to elucidate th e dynamics of phytoparasitic nem atode populations. M. hapla is an appropriate species for quantitative population studies due to its residency within root tissue, discernible life stages, and habits of oviposition (e.g., eggs are contained in a single unit, or egg mass). The shallow-rooted and nonfibrous root system of A. cepa contributes to this plant's usefulness as a "laboratory rat" for nematological research. R oot-knot nem atodes are only one component among many biological and environm ental factors influencing the growth and yield of plants in agroecosys­ tem s. The dynamics of Meloidogyne populations are influenced by clim atic conditions and soil biota surrounding th eir plant hosts. Previous studies have examined th e im pact of abiotic facto rs such as tem perature (Wong and Mai, 1973a), moisture (Wong and Mai, 1973b), and edaphic param eters (Wallace, 1969) on the ontogeny of Meloidogyne spp. The influence of heritable a ttrib u tes such 5 as nematode reproductive capacity (Ferris and Stuth, 1982; Tyler, 1938) and host quality (Balasubramanian and Rangaswami, 1963) on th e increase of Meloidogyne populations has also been addressed, and the role of concom itant soil organisms in determ ining nem atode population change has received some atten tio n (Sayre, 1980). The relationship between M, hapla and other members of the soil communi­ ty is an appropriate and tim ely research topic. E stim ates of plant damage attributable to root-knot nem atodes in the presence of organisms beneficial or pathogenic to the host crop are essential for the accu rate prediction of crop losses. More im portantly, the identification and nurture of natural biological controls for nem atode pests will become a crucial agricultural management activ ity as the cost of pesticide application increases. There is a distinct need for alternatives to th e many pesticide-dependent programs for nematode control. Prices of existing nem aticides are rising yearly, and the cost to develop new pesticides increased three-fold from the 1960s to the 1970s (Edens, 1978). The efficacy of pesticides is more carefully scrutinized now ■than in the past. For exam ple, according to Rohde e t al. (1980), the use of fumigant biocides can lead to the eventual increase of Meloidogyne populations to levels equal to th a t of unfumigated areas. Although fungi, bacteria, soil invertebrates, and other nem atodes have been reported to influence th e increase of Meloidogyne populations, attem p ts to utilize knowledge of nematode interactions with soil biota for nem atode manage­ m ent are few. The scarcity of such programs is due, in p a rt, to an insufficient understanding of the role of these soil organisms in agroecosystem s. Of all possible candidates for the biocontrol of nem atodes, VAM are unique in th a t not 6 only have they been Im plicated as a facto r im pacting nem atode population growth, but are them selves th e subject of extensive study. The potential benefits of research investigating the interaction between the increase of M. hapla, VAM, and plant growth are impressive. VAM colonization increases the uptake of phosphorus and other soil nutrients, thus stim ulating plant growth. Drought tolerance is also increased in m ycorrhizal A. cepa (Nelsen and Safir, 1982a; 1982b). Added to these benefits, evidence of the suppression of nem atode populations by VAM would support th e im plem entation of management schemes to prom ote mycorrhizal developm ent in agricultural production system s. The e ffe c t of VAM on the population dynamics of root-knot nem atodes has been addressed in several studies, although many aspects of the nematode-fungus relationship remain unanswered. Sikora (1979) proposed four ways th a t mycor­ rhizal fungi influence plant-parasitic nem atodes: (1) altering root attractiv en ess, (2) reducing larval penetration, (3) impeding larval developm ent, and, (4) retarding giant cell form ation. Three additional means of decreasing nem atode numbers can be added to this list: (1) reducing nematode survival, (2) altering sex ratios, and (3) reducing fecundity. No study to date has thoroughly examined these alternatives. Sikora and Schonbeck (1975) are credited with dem onstrating the influence of VAM on the penetration and developm ent of Meloidogyne juveniles. Two experim ents are reported in th eir paper, one lasting 30 days (one harvest) and one lasting 125 days (harvests a t 42, 84, and 126 days). N either experim ent successfully differentiated the contribution of decreased penetra­ tion, m ortality within roots, and ra te s of development and reproduction to nematode loss in mycorrhizal roots. 7 The e ffe c t of VAM on nematode survival within roots or soil has not been examined. No study has attem p ted to enum erate d ifferen t juvenile stages of MeloidoRvne in m ycorrhizal roots. Gall indices or counts of eggs, juveniles, or adult nem atodes have been used in previous studies. The presence of VAM was dem onstrated to retard or inhibit giant cell form ation (Sikora, 1979). probable, therefore, th a t VAM contribute to nem atode m ortality. It is In fa c t, decreased nem atode densities in several experim ents could have been due to nem atode m ortality rath er than to the delayed developm ent of nem atodes, as was interpreted from the data. In addition to providing knowledge concerning ecological associations influencing the population dynamics of nem atodes, aspects of this research have potential application for the production of muck vegetables in Michigan. M. hapla is a prevalent plant-parasitic nem atode in Michigan onion acreage and has long been recognized as a serious pest of carro t and celery. The im portance of M. hapla as a pathogen to onion has not been adequately assessed, nor has the relative effectiveness of onion in m aintaining populations of _M. hapla been characterized. The population dynamics of nem atodes is a complex phenomenon and cannot be properly assessed by th e reductionist approach adopted so frequently in past studies. To truely understand the activ ities of nem atodes or other pests, it is necessary to m aintain a holistic conception of the pest, crop, and other interacting components of the agroecosystem . C onstraints of tim e, facilities, and manpower d ic ta te th a t th e pest-crop agroecosystem be experim entally examined piecem eal. It is im perative, however, th a t th e research is designed and analyzed w ithout losing sight of the interdependence betw een all components 8 in the ecosystem . The complexity and intricacies of the association between organisms and their environment has too often led to the perception th a t th e holistic approach is inappropriate and unrealistic for biological system s. Fortu­ nately, the advent of high-speed com puters and system s science methodology, developed by engineers to deal with com plexity in th e physical sciences, shows this perception to be false. Systems science is a problem solving procedure to evaluate and make decisions concerning the stru ctu re, function, and behavior of complex system s. A system is defined as a "regularly interacting or interdependent group of item s forming a unified whole"* and so this methodology can be applied to any system where the elem ents involved can be identified and th eir interrelationship conceptualized. The systems approach has been widely applied in studying m echanical, e lec trica l, chem ical, and other physical system s and has recently been used to examine economic, social, and biological system s. Im plicit to the system s approach is the consideration of all factors th a t contribute to the solution of problems presented by th e behavior of th e system — e.g., holism. Inclusion of all factors in the problem-solving process leads to the realization th a t "trade-offs" in benefits to individual components of th e system are inevitable. In the onion agroecosystem , for. exam ple, fumigant nem aticides applied to reduce nem atode populations may also be d etrim ental to beneficial organisms such as nematophagous insects or VAM. There are many advantages to using system s methodology when dealing w ith nem atodes or other pests. No one person can deal with the diversity inherent to biological systems, so th e effo rts of a transdisciplinary team must be *W ebster's New C ollegiate D ictionary, 1976. 9 coordinated to solve the problems of increased crop production. The activities of all involved researchers are organized by th e logic and methodology of system s science. A holistic approach is conducive to long-term planning and solutions to pest problems th a t are ecologically sound. Most im portantly, th e use of systems methodology has led to the development of flexible, economic pest managem ent strateg ies (on-line pest management) and the realization th a t solutions to pest problems may lie in the redesign of the stru ctu re of agroecosys­ tem s (Edens and Haynes, 1982; Tummala and Haynes, 1977). An established hierarchy of activ ities is inherent to the system s approach. The initial phase in the process is a feasibility analysis which includes a definition of needs, the form ulation of the problem one wishes to solve, and the generation and exam ination of alternative ways to redesign th e stru ctu re of the system to m eet the desired needs. Next, an abstraction of the system in the form of analytical or com puter simulation models is developed to analyze the behavior of the system . For complex system s, models representing only a portion of the system can be developed and la te r in terfaced together so th a t the e n tire system is represented. This technique, referred to as the discrete component approach (Tummala e t al., 1975), is particularly useful for biological system s. Finally, the details of the system stru ctu re and m anagem ent strategies identified earlier are operationalized and evaluated in th e real world (Allen and Bath, 1980). The identification of th e system is an insightful and valuable activity for the biologist. The "object of control", identified in the form ulation of needs, determ ines those components or "system design param eters" to be included in the system and those considered to be exogenous to the system (hereafter 10 referred to as the environment). Environmental variables influencing the system can be described as controllable (e.g., plant cultivar) or uncontrollable (e.g., w eather). The behavior of the system can be assessed by measuring desired (e.g., decreased nem atode density) and undesired (e.g., overcrowding of plants) system output variables. To illustrate these concepts, th e M. hapla-onion system is identified in Figure 1. Components other than M. hapla are characterized according to their level of involvement in the population dynamics of this nem atode. BACKGROUND Life History Meloidogyne hapla progresses through six developm ental stages in the course of its lifetim e: egg, four juvenile stages, and adult. Eggs are deposited into a gelatinous m atrix extruded from specialized glands of the fem ale. Individual eggs are retained in this egg mass, which adheres to th e root surface or lays amid soil particles. Within the egg, the embryo develops into a first- and then a second-stage juvenile. Specific environm ental stimuli elicit m echanical and chemical responses from the second-stage juvenile th a t in itiate egg hatch (Bird, 1971). The second-stage juvenile moves through the soil in search of a host plant. Movement of th e juvenile is not random, but rath er is directed by stimuii associated with a developing plant (Green, 1971). Piercing the root with its sty let, th e nem atode m igrates intercellularly in th e root cortex in search of an appropriate feeding site. Hyperplastic responses of the infected plant, hyper­ trophy and hyperplasia, can be d etected soon a fte r the juvenile begins feeding. Monitored Environment R eferen ce T 1 Point T O rder I 1 Order II ABIOTIC F ACTORS Temperature M yc o rrhizae Moisture Soil N u t r i e n t s pH I BIOTIC F A CTOR S R o ot-K n o t Nem atode Onion I Predaceous Onion Maggot N ematodes Phytoparasitic N ematodes Figure i . R o o tLesion Nematode C o n c e p tu a li z a ti o n o f th e hi. hapla - A. cepa ecosystem with M. hapla as th e r e f e r e n c e p o i n t . 12 Disagreement arises in the lite ra tu re as to the sequence of developm ental changes occurring for root-inhabiting juveniles. According to Nagakura (1930) and R itte r and R itter (1938), nem atodes m olt from the second stage to the third stage soon a fte r feeding commences. O ther authors (Triantaphyllou and Hirschmann, 1960; Bird, 1939) report th a t considerable growth of th e second-stage juvenile and sexual differentiation occur before th e m olt to the third stage. The la tte r view is generally accepted. An extim ated 30 - 70% o f the tim e spent in the developm ent of M. hapla from root penetration to oviposition is passed within the second juvenile stage (Ferris and Hunt, 1979; Triantaphyllou and Hirschmann, 1960; Tyler, 1933; Vrain e t al., 1978; Wong and Mai, 1973a). The third and fourth juvenile stages occur in relatively rapid succession. The cuticle of the second stage is retained as th e juvenile m olts to the third stage. Similarly, both the second- and third-stage cuticles are retained in the fourth-stage juvenile. Third- and fourth-stage juveniles do not feed as a result of this cuticular encasem ent and a degeneration of the sty le t, although m atura­ tion continues. Fem ales retain a saccate shape throughout the developm ental period, but males undergo a m etamorphosis in the fourth juvenile stage to the eelworm shape of soil-inhabiting nem atodes. Following a fourth m olt, the adult fem ale resum es feeding and increases substantially in size. The adult male does not feed and a fte r m ating m igrates away from the root system . There is a considerable delay between the m olt to adulthood and commencement of oviposition of the fem ale. Approximately 30% of the tim e necessary for development of the fem ale, from root penetration until the appearance of eggs, is passed as a preovipositing adult (Bird and Wallace, 1965; Wong and Mai, 1973a). 13 M. incognita fem ales produce ca 150 - 500 eggs per egg mass (Carlson and Rothfus, 1978). Tyler (1933), working m ost probably with M. hapla (Thomason and L ear, 1961), estim ated th a t one egg was produced per 12 degree hours (base 10 C). The duration of the oviposition period of M. hapla has not been reported, but according to Carlson and Rothfus (1978) M. incognita fem ales rem ain alive and lay eggs through a t least one additional generation. The age of th e fem ale does not seem to influence egg production; de Guiran and R itte r (1979) observed no differences in th e weekly ra te or to ta l egg production of M. incognita over a four-w eek period. Factors determ ining the ratio of fem ales to males is not well understood. No sex chromosomes have been observed in plant-parasitic nem atodes and sexual differentiation in Meloidogyne spp. seem s dependent on environm ental facto rs. Under conditions favorable to nem atode developm ent (suitable tem p eratu re, m oisture, host, e tc.) all or most juveniles develop into fem ales. Under unsuitable conditions, males as well as fem ales occur. The sex of M. hapla and other Meloidogyne spp. is determ ined in the second juvenile stage. Second-stage juveniles developing as fem ales may rev ert to males if unfavorable conditions arise before the second-stage is com pleted. U ndifferentiated juveniles which become m ales, and fem ales which undergo sex reversal are referred to as "true males". In M. javanica male intersexes, i.e., males with fem ale sex ch aracter­ istics, also occur. The role of Meloidogyne males in reproduction is variable among the species. No obligatory am phim ictic species have been described for this genus. M. hapla populations show variation in chromosome number and mode of reproduction. Populations described as R ace A reproduce by amphimixis, or \k m eiotic parthenogenesis, or both. Populations described as R ace B reproduce by m itotic parthenogensis (Triantaphyllou, 1966). The number of generations of M. hapia occurring per year depends on the host crop and clim atic conditions. In Michigan muck soils two generations are observed in an average year, with peak nem atode densities evident in early August. M. hapla overw inters in the egg stage. Several adaptations enable M. hapla to survive conditions unsuitable to nem atode developm ent. Eggs retained within the protective covering of th e gelatinous m atrix successfully w ithstand freezing tem peratures. Many nem a­ todes, probably including M. hapla juveniles, exhibit decreased m etabolic activ ity when exposed to clim atic extrem es (Cooper and Van Gundy, 1971). Quiescent states for many nem atode genera are evoked by low soil m oisture, oxygen, and tem perature. In addition, cryptobiosis, th e alm ost com plete shutdown of m etabolism , ailows nem atodes to survive long periods of low soil m oisture or tem perature. M ortality ra te s of M. hapla due to biotic, abiotic, and human factors have not been clearly defined. Examination of u ata from several studies (Dropkin, 1963; Griffin and Elgin, 1977; Wong and Mai, 1973b) indicate th a t ca 38% of M. hapla eggs eventually become root-inhabiting nem atodes. E stim ates of the m ortality of juveniles within roots are not available. Pathogenicity of Meloidogyne hapla JM. hapla has a wide host range, including crop plants and weeds. General symptoms of all plants to infection by Meloidogyne spp. include stunting, yellowing, and the proliferation of lateral roots. The m ost obvious symptom of 15 root-knot infection is root galling, although galls formed by M. hapla are usually sm aller than those produced by th e other most common Meloidogyne spp. Pathogenicity of M. hapla varies with th e host crop, but this nematode is generally considered to be a detrim ent to plant growth and yield (Franklin, 1979). Meloidogyne infection induces m echanical and physiological changes th a t can a lte r th e relationship betw een some plants and other pathogens. Parasitism of plants by IW. hapla may increase the incidence and severity of both fungal (Jenkins and Coursen, 1957; Meagher and Jenkins, 1970) and b acterial (Griffin e t al., 1968; Hunt e t al., 1971) diseases. Moreover, the resistance of plants to fungal disease can be broken by root-knot infection (Sidhu and Webster, 1977). L ittle is known concerning the relationship between nem atodes, insects, and plant growth, although it has been hypothesized (Wallace, 1973) th a t nem atode infection might influence the susceptibility of a plant to insect atta ck . Onion, Allium cepa L., is listed as a host for M. hapla, M. incognita, and M. javanica (Chitwood, 1951; Martin, 1961; Tyler, 1933). Some reports indicate th at economic losses of onion may result from M. hapla infection (Kotcon, 1979; Olthof and P o tter, 1972; Sherf and Stone, 1956; Smith, 1964). According to other sources (Chitwood, 1951; Tyler, 1933) onion is relatively resistant to M. hapla and only lim ited development of nematodes and resulting yield losses occur. Discrepencies in th e lite ra tu re may be due to variability in the damage and reproduction of M. hapla associated with d ifferen t onion cultivars (Franklin and Hooper, 1959; Kotcon, 1979; Sasser, 1954). The relative effectiveness of onion in maintaining populations of M. hapla has not been definitively characterized. Chitwood (1951) found th a t M. hapla was unable to reproduce on Downing Yellow Globe onions. Observations of M. 16 hapla reproduction on Yellow Globe (Kotcon, 1979; Lewis e t al., 1958; Olthof and P o tte r, 1972) and A ristocrat (Smith, 1964) varieties were published subsequent to Chitwood's report. These studies, conducted under laboratory conditions, did not determ ine if M. hapla levels can increase on onion to the point where succeeding onion crops are damaged under field conditions. Interaction Between Meloidogyne hapla and A biotic F actors Tem perature The e ffe c t of tem perature on Meloidogyne spp. has received much a tte n ­ tion. The range of optim al tem perature differs betw een species (Bird and Wallace, 1965; Dropkin, 1963; Vrain and Barker, 1978). therm al optima than do m ost other species. M. hapla has lower D ifferential growth responses to tem perature have also been reported between populations of M. javanica (Daulton and Nusbaum, 1961) and between races of M. naasi (Michell e t al., 1973). Although variability in therm al tolerance has not been tested for M. hapla. a comparison of the results reported by Wong and Mai (1973a) in New York and Vrain e t al. (1978) in North Carolina suggest th a t such differences do exist. Among populations of Meloidogyne spp. differential e ffe cts of tem perature on each life stage have been noted (Bird and Wallace, 1965; Wallace, 1971). In general, soil-inhabiting stages have lower tem perature ranges than do the rootinhabiting stages. A similar phenomenon has been reported for H eterodera schachtii (Wallace, 1973). Tem perature affects all life stages of Meloidogyne spp. Eggs of M. hapla hatch optimally a t ca 20 C (Vrain and Barker, 1978). Wuest and Bloom (1965), however, found th a t the therm al optim a for M. hapla eggs decreased with tim e. 17 The tem perature for the maximum survival of eggs incubated three and 30 days was 27 C and 12 C, respectively. Tem perature influences the movement of M. hapla juveniles in soil and the ability of juveniles to invade plant roots. Bird and Wallace (1963) found th a t the movement of juveniles through a sand column was fa stest a t 20 C and mobility was severly lim ited by tem peratures of 30 and 35 C. Consequently, these authors observed th a t significantly g reater numbers of juveniles penetrated roots under a 15 - 20 C tem perature regime than did juveniles reared under 20 - 25 C and 25 - 30 C regimes. Vrain and Barker (1978) dem onstrated th a t the minimum tem perature for root penetration by M. hapla is 8.8 C. Development of M. hapla juveniles inside roots proceeds a t tem peratures as low as 12 C (Vrain e t al., 1978) and as high as 32.3 C (Wong and Mai, 1973a). The optimal tem perature for juvenile development is ca 25 C (Wong and Mai, 1973a). H eat units are generally used to re la te tem perature and developmental tim e. One heat unit (= one degree hour) equals one degree above the developmental threshold acting for one hour. Based on a developmental threshold of 10 C (data w ere adjusted where necessary) estim ates of ca 9500, 6500, 7500, and 8000 heat units were reported by Wong and Mai (1973a), Starr and Mai (1976), Vrain e t al. (1978), and -Tyler (1933), respectively, as the developm ental tim e necessary for second-stage juveniles of M. hapla within roots to become egg-producing fem ales. An additional period of 4000 - 5000 heat units is required for egg developm ent and hatch (Tyler, 1933). The above estim ates are similar to those reported for other Meloidogyne spp. (Bird and Wallace, 1965; Ferris and Hunt, 1979; Milne and Duplessis, 1964). It should be noted th a t these estim ates of M. hapla development are based on th e first appearance of eggs and 18 do not represent the average developmental tim e of all observed nem atodes. Considerable variation between the developm ental ra te s of th e fa stest and the average nem atode occur (Ferris and Hunt, 1979). Tem perature is one of the facto rs influencing sex determ ination of Meloidogyne spp. Davide and Triantaphyllou (1967) found th a t a t tem peratures of 20 - 35 C only one percent of M. incognita reared on tom ato developed into m ales while a t 15 C, 6.7% developed as m ales. The hypothesis th a t differential m ortality between the sexes rath er than sex expression was responsible for the results was rejected, because no dead juveniles were observed. Laughlin e t al. (1969) reported even higher m ale/fem ale ratio s for M. graminis reared under high tem peratures; a t 16 and 21 C, no males were observed; a t 27 C, 4% of the nem atodes were males; a t 32 C, 80% of all nem atodes developed as m ales. No inform ation is available regarding the e ffe c t of tem p eratu re on the sex determ i­ nation of M. hapla. The optim al tem perature for reproduction by M. hapla is from 25 - 30 C (Griffin and Elgin, 1977; Thomason and Lear, 1961). The maximum and minimum tem perature for reproduction by this species on tom ato are 32.6 and 15.0 C (constant tem perature), respectively. Wong and Mai (1973a) reported th a t under a tem perature regime of 21.1 - 26.7 C, 1662 eggs and juveniles per gram of root were produced by M. hapla infecting le ttu c e. During th e sam e tim e span, under a higher tem perature regime of 26.7 -32.2 C, 3019 eggs and juveniles per gram of root were produced from th e initial inoculum of 400 juveniles/plant. Meloidogyne spp. differ in their susceptibility to high and low tem perature extrem es. M. hapla can survive lower tem peratures than M. javanica or M. incognita (Bergeson, 1959; Daulton and Nusbaum, 1961; Vrain e t al., 1978). 19 Conversely, M. hapla are less able to survive high soil tem peratures (Daulton and Nusbaum, 1961). Several factors influence the ability of M. hapla to withstand freezing tem peratures. Longer exposure tim es to low tem peratures are necessary to kill eggs in dry soil than in w et soil (Daulton and Nusbaum, 1961). The stage of the nem atode a ffe cts survival ability. Unhatched juveniles survive freezing tem per­ atures b e tte r than the embryonic stages within eggs of M. hapla (Vrain e t al., 197S). The habitat of th e nem atode is also im portant. All stages of M. hapla survive freezing tem peratures when protected by root tissue (Vrain e t al., 1978). The tolerance of M. hapla to freezing indicates the presence of a mechanism for withstanding low tem peratures (Sayre, 1964). There is evidence th a t an acclim ation period to low tem perature enhances survival. Nusbaum (1962) found th a t egg masses buried outdoors in August show g reater survival th e next spring than do egg masses buried in November. Moisture Soil-inhabiting nem atodes are essentially aquatic organisms. Soil m oisture directly influences the developm ental ra te s of Meloidogyne eggs and infectivity of second-stage juveniles (Ogunfowora, 1978; Wallace, 1968b; Wong and Mai, 1973b). W ater provides a medium for movement of nem atodes within th e soil. D ifferences in tolerance of soil m oisture stress by geographically isolated populations of Meloidogyne spp. occur (Daulton and Nusbaum, 1961). tem perature, soil moisture content and resulting As with concentrations have differ­ ential e ffe c ts on each stage of nem atode developm ent (Godfrey and M orita, 1929; Wallace, 1966; 1968a). 20 Soil moisture influences the ra te of development and hatch of Meloidogyne eggs. Decreased O j levels associated with high soil moisture conditions a ffe c t developing embryos more severely than juveniles within eggs of M. javanica (Wallace, 1968a). Egg m ortality results if embryonated eggs a re subjected to anaerobic conditions for one week (Ferris and Van Gundy, 1979). The e ffe c t of O 2 on egg hatch can be mediated by corresponding CO 2 levels and it may be the ratio of O j/C O j rather than C>2 aione th a t is responsibie for the inhibition of egg hatch (Wong and Mai, 1973b). Egg development and hatch are also decreased under low soil moisture conditions. Dehydration of eggs seems to a ffe c t secondstage juveniles more severly than developing embryos and first-stag e juveniles, due to the increased permeability of the egg shell prior to hatch (Wallace, 1966). Optimal egg hatch and development occurs when the soil moisture level is a t field capacity. Meloidogyne eggs exhibit mechanisms for surviving low soil moisture extrem es. High osmotic concentrations, simulating moisture stress, inhibit the hatch of M. arenaria eggs, although exposure to even high concentrations (1M solutions of NaCl, KC1, C a C ^ or dextrose) for 33 days does not prevent hatch when eggs are transferred to w ater (Dropkin e t al., 1938). Soil moisture levels have a g reater e ffe c t on the activity of M. hapla juveniles in th e soil. No penetration of lettu ce roots occurs in w ater-saturated soil (Wong and Mai, 1973b) and flooding has long been used to reduce nematode populations (Dropkin, 1980). Low soil moisture impedes nematode movement due to the redistribution of the w ater surrounding soil particles. Soil m oisture levels unfavorable to nem atode activity m ost often delay rather than prevent infection. The e ffe ct of soil m oisture on the root-inhabiting stages of Meloidogyne and other endoparasitic nematodes is not known. The influence of soil moisture 21 on nematodes reproducing within plant roots is certainly m ediated through the host plant. It has been observed th a t decreased plant vigor limits reproduction by Meloidogyne spp., although the specific e ffe cts of w ater stress on this phenomenon a re not known. Soil texture Soil tex tu re and composition a ffe c t th e tem p eratu re conductivity and m oisture relations of the soil environm ent. The size of soil particles determ ines the number and size of pore spaces and consequently, the ability of nematode juveniles and w ater to move through the soil. Both very small and large pore spaces tend to inhibit Meloidogyne movement. Invasion and reproduction of M. javanica infecting tom ato is g re a ter in fine sand (150 - 250 u) than in coarse (500 - 600 u) or medium (250 - 500 u) sand (Wallace, 1969). Soils with many pore spaces, however, lim it the reproduction (as measured by th e number of secondstage juveniles recovered from soil) of M. hapla infecting sugar beet (Santo and Bolander, 1979). Soil pH The e ffe c t of pH on the hatch of Meloidogyne eggs and invasion of roots by second-stage juveniles appears to be minimal (Watson and Lownsbery, 1970). Lowenberg e t al. (1960) found th a t the optim al pH for egg hatch and survival of M. incognita was 6.4 in H eller's medium. It seems likely th a t the range of pH found in most agricultural soils is favorable for the development of the soilinhabiting stages of Meloigodyne spp. Once infection has occurred, the influence of pH on nematode developm ent is probably m ediated through the host plant. 22 Interaction Between Meloidogyne hapla and Biotic Factors Predators and parasites The association between plant-parasitic nem atodes and other soil organ­ isms has often received low priority in nematology research programs. Conse­ quently, prim arily descriptive accounts of interactions between nem atodes and their predators and parasites have been published. The egg and second juvenile stages of M. hapla are susceptible to predation by a variety of organisms (Esser and Sobers, 1964; Sayre, 1971; 1980). Preda­ ceous fungi, ubiquitous in most soils, trap Meloidogyne and other nem atodes by specialized mechanisms such as adhesive networks, sticky knobs, or constricting hyphal rings (Barron, 1977). Some predaceous nematode genera including Mononchus. Mononchoides. Buterius, Anatonchus, Diplogaster, Tripyla, Seinura, and some species of Dorylaimus, Discolaimus. and Actinolaimus also prey upon Meloidogyne (Esser and Sobers, 1964). O ther in v erteb rate predators of nema­ todes include tardigrades (Doncaster and Hooper, 1961), tubellarians, m ites, protozoans, oligochaetes, and Collembola (Gilmore, 1970). The soil-inhabiting stages of M. hapla are parasitized by several organisms (Sayre, 1971). C ertain endozoic fungi parasitize Meloidogyne spp., including D actylella (Stirling and Mankau, 1978), Haptoglossa (Esser, 1976), and C atenaria (Birchfield, 1960). Only one report of a virus disease in nem atodes (M. incognita) exists in the literatu re (Sayre, 1971). The nem atode parasite receiving the most attention has been the bacteria Bacillus penetrans (earlier classified as a protozoan). Bacillus penetrans has been identified infecting Meloidogyne spp. (Sayre and Wergin, 1979) and work investigating th e viability of this pathogen as a biological control agent is now in progress. 23 Other phvtoparasitic nematodes The increase of M. hapla is related to nem atode density. Low density levels are inhibitory to populations in which reproduction depends on the location of the opposite sex (Seinhorst, 1968). High nem atode density inhibits root colonization and leads to com petition for food reserves or feeding sites. Damage by nem atodes reduce plant growth, thereby lim iting th e number of nem atodes the plant can support (Seinhorst, 1967). The population dynamics of M. hapla is also influenced by other nematode genera. As discussed previously, predatory nem atodes may reduce levels of th e soil-inhabiting stages. Com petition with other root-inhabiting genera of Meloid­ ogyne spp. a ffe c ts levels of M. hapla within roots (Chapman, 1966; Estores and Chen, 1972; Gay and Bird, 1973). Inter- and intra-specific com petition regulate the penetration of roots by M. hapla juveniles. Decreased infectivity of M. hapla was observed in roots simultaneously inoculated with M. javanica and the penetration of tom ato roots by M. hapla is negatively correlated with inoculum level (Kinloch and Allen, 1972). Conversely, the penetation of tom ato roots by JW. javanica is not density dependent. Kinloch and Allen (1972) hypothesized th a t in contrast to M. javanica. M. hapla is not a ttra c te d to, or cannot p en etrate galled tissue. This hypothesis is supported by the higher incidence of term inal root galls and th e lower mean number of cohabiting nem atodes per gall for this species. Pratylenchus spp. (migratory endoparasitic nematodes) can also inhibit root penetration by root-knot nem atodes. Simultaneous inoculation or previous inoculation of P. penetrans (Turner and Chapman, 1972) or P. brachyurus (Gay and Bird, 1973) decreased root penetration by M. incognita. Penetration by 24 Pratvlenchus was unaffected in both studies. In the presence of M. incognita. numbers of P. brachyurus were increased on cotton, decreased on tom ato, and unaffected on alfalfa or tobacco. The Gay and Bird study illu strates the complexity of interactions between nem atode species. The e ffe c t of nematode density and mixed populations on the development of M. hapla within roots has received little atten tio n . Studies using several Meloidogyne spp. are impeded by the difficulty of identifying pre-adult stages to the species level. The e ffe cts of intra-specific com petition on nem atode development are more easily discernible. Davide and Triantaphyllou (1967) found th a t high infection density reduced the ra te of developm ent of incognita and M. javanica. Wallace (1969) attrib u ted the decrease of M. javanica adult fem ales a fte r 20 days with increasing inoculum levels, to delayed developm ent resulting from com petition for feeding sites. His conclusions were based on findings th at the ra te of root invasion was unaffected by population level; root growth was unaffected by population density; th ere was no indication of d ifferential m ortali­ ty; and the e ffe ct disappeared in plants reared for 42 days. Decreased reproduction by root-knot nem atodes a t high densities or with concom itant infection of other Meloidogyne or Pratylenchus spp. has been noted. A correlation between decreased egg production by M. javanica infecting tom ato and inoculum level was observed by Wallace (1969). The reproduction of M. hapla on tobacco was reduced by the presence of M. incognita or P. brachyurus (Johnson and Nusbaum 1970). Greenhouse tem peratures in this study may have been less favorable for M. hapla and thus partially responsible for th e reported e ffe c t. Again, the complexity and difficulty of assessing nem atode-nem atode interactions is illustrated. 25 High nem atode density can also alter th e sex ratios of some Meloidogyne spp. Davide and Triantaphyllou (1967) found th a t inoculum levels of 350 and 50,000 M. incognita juveniles per tom ato plant resulted in populations with 0.5% and 53.5% m ales, respectively, although high nem atode density had no e ffe c t on the proportion of males in populations of M. javanica. Vesicular-arbuscular mycorrhizae The beneficial e ffe cts of vesicular-arbuscular m ycorrhizae (VAM) on plant growth are well documented (Atilano e t al., 1981; Bird e t al,, 1974; Hussey and Roncadori, 1978; Rich and Bird, 1974). Stim ulation of plant growth due to a VAM association has been attrib u ted to increased w ater and nutrient uptake and a reduction of pathogenic organisms. Several reports indicate th a t mycorrhizal colonization negates the deleterious e ffe c ts of nem atodes on plant growth (Kellam and Schenck, 1980; Hussey and Roncadori, 1978, 1982; Roncadori and Hussey, 1977). The mechanisms by which VAM lim it nem atode damage to plants has not been determ ined. The hypothesis tested most often in previous studies is th a t nem atode density, and hence damage, is reduced as a consequence of VAM colonization. Kellam and Schenck (1980) found th a t M. incognita produced few er galls on mycorrhizal soybeans, both on a per plant and per gram of root basis. Bagyaraj e t al. (1979) also observed fewer galls on tom ato plants jointly inoculated with M. incognita or M. javanica and Glomus fasciculatus (VAM) than on plants inoculated with the nem atode alone. A decrease in the number of root-knot nem atodes infecting mycorrhizal tom ato, tobacco, o a t, and carro t was reported by Sikora and Schonbeck (1975). It should be noted, however, th a t differences in 26 nem atode levels between mycorrhizal and non-mycorrhizal plants decreased tow ards th e end of the Bagyaraj and Sikora studies. Conversely, several authors concluded th a t VAM colonization enhances the growth of nem atode populations (Atilano e t al., 1981; Kotcon, 1979; Roncadori and Hussey, 1977; Schenck e t al., 1975). Roncadori and Hussey (1977) ex tracted m ore M. incognita eggs from m ycorrhizal cotton than from non-mycorrhizal plants. The same authors (Hussey and Roncadori, 1978) observed th a t mycorrhiz­ al cotton supported g reater numbers of Pratylenchus brachyurus than did nonmycorrhizal plants. In both studies, however, mycorrhizal roots supported fewer nem atodes on a per gram unit basis. A positive correlation betw een the density of Meloidogyne spp. and VAM colonization has also been observed on onion (Kotcon, 1979), soybean (Schenck and Kellam, 1978), and grape (Atilano e t a l., 1981). D ifferences in the methodology of previous experim ents may account for inconsistant results regarding the nematode-VAM interaction. The association between root-knot nem atodes and VAM is dependent on th e host plant, levels of nem atode and fungal inoculum, and th e tim ing of inoculation (Schenck, e t al., 1975). More im portantly, exam ination of d ata from previous studies shows th a t th e interaction between plant, nem atode, and VAM is dynamic. Time has been considered as a variable in only four studies (Bagyaraj e t al., 1979; Kellam and Schenck, 1980; Kotcon, 1979; Sikora and Schonbeck, 1975). In two of these studies (Bagyaraj e t al., 1979; Kellam and Schecnk, 1980) gall indices rath er than nem atode numbers were recorded. Galling does not necessarily re fle c t popula­ tion levels of M. hapla, since the number of nem atodes inhabiting each gall is variable (Kinloch and Allen, 1972). Of th e remaining studies, only adults were 27 measured in one experim ent (Sikora and Schonbeck, 1975) and the other experim ent was biased by very poor plant growth (Kotcon, 1979). L ittle is known concerning th e influence of VAM on the ontogeny of M. hapla and other plant-parasitic nem atodes. It has been hypothesized th a t fewer M. hapla juveniles penetrate mycorrhizal roots (Sitaram aiah and Sikora, 19S0). Root e x tra c ts from mycorrhizal roots, however, do not repel nem atodes (Sikora and Schonbeck, 1975). It has been speculated th a t VAM alter the physiology of the plant making i t less susceptible to nem atode a tta c k (Bagyaraj e t al., 1979; Sikora, 1979) or com pete with nem atodes for colonization sites within the root (Fox and Spasoff, 1972; Kellam and Schenck, 1980; Hussey and Roncadori, 1978). The development of root-knot nem atodes seems to be impeded in mycorrhizal tissue. B altruschat e t al. (1973) reported a 75% reduction in th e number of M. incognita juveniles th a t developed into adults on m ycorrhizal tom ato. Similarly, Sikora and Schonbeck (1975) observed fewer adult M. incognita on mycorrhizal tobacco, o at, and tom ato than on nonmycorrhizal plants a fte r 30 days. These authors report similar findings for M. hapla infecting carro t. Only tw o studies have examined th e influence of VAM on nematode reproduction. Roncadori and Hussey (1977) found g reater numbers of M. incognita eggs on mycorrhizal cotton plants m aintained under low or high fe rtility conditions. Calculations of eggs per g of root tissue, however, showed th a t egg numbers were lower on mycorrhizal plants grown under low fertility conditions, compared to non-mycorrhizal controls, due to differences in th e size of the root system s. These authors did not count the number of adult fem ales responsible for egg production. The influence of Glomus mosseae on the fecundity of Rotylenchulus reniform is was examined by Sitaram aiah and Sikora 28 (1980). Adult fem ales produced fewer egg masses and laid few er eggs per egg mass on mycorrhizal tom ato. Conceptual Model of Meloidogyne hapla Associated with Allium cepa D iscrete component models of Meloidogyne hapla and other components of the onion agroecosystem were constructed by myself and other researchers a t Michigan S ta te University. My objectives in developing th e M. hapla model were to: (1) assim ilate inform ation previously published on the biology of M. hapla. (2) identify inform ation gaps concerning the interaction betw een M. hapla and A. cepa. (3) develop a m athem atical description of the population dynamics of M. hapla th a t could incorporate data from my research projects to an existing data base, and (4) provide a means for comparing laboratory and field d ata. The model sim ulates the population dynamics of M. hapla associated with a single onion plant. The daily development of seven nem atode life stages is predicted by the model according to soil tem perature, using inform ation from studies cited previously. Other abiotic factors such as soil m oisture, tex tu re, nutrient levels, and pH are not currently included in th e model, due to a lack of quantitative data, but can be added when an appropriate data base is developed. Similarly, little inform ation on the relationship betw een M. hapla and the growth of A. cepa was available when the model was first developed. D ata from my experim ents were used to describe the influence of M. hapla on th e growth of A. cepa and to estim ate the amount of root substrate available for nematode ingress and nourishment. 29 Overview of the Model Figure 2 shows a simplified diagram of the M. hapia model. The nem atode population is divided into egg, juvenile, and adult stages. Juveniles are fu rth er characterized according to a soil or root hab itat. Flows from one stage to another are dictated by tem perature-dependent developm ental ra te s and by constraints imposed by the onion plant. Onion root growth determ ines the substrate available for nem atode penetration. Nem atode feeding inhibits root growth, thereby lim iting the number of nem atodes eligible for root penetration. A flow chart of the model is presented in Figure 3. The user is prompted for initial egg density, planting d ate, length of the growing season, th e tim e increm ent for the simulation, and w hether or not onion plants have a VAM association. Field soil tem perature is read from a tape a t each daily iteration. Degree hour (base 9 C) accum ulation is computed and the various subroutines are called. Model output is stored a t the end of each daily iteration. Description of Subroutines Subroutine Onion (Figure 4) A simple regression model of onion root growth was substituted for a more complex onion plant model. Presently, root grow th from planting until 34 days a fte r planting is described by the equation: 0.0095537 * e 0*0924842 * numday * q j (numday = day since planting) and from 54 days a fte r planting until harvest by th e equation: 0.000002694 * numday3*340064 * DT This value for root growth under optim al conditons is then modified by tem perature according to the function: TEMPERATURE i EGGS m r SOIL ROOT LARVAE OO U LARVAE ADULT FEMALES ONION ROOT WEIGHT Figure 2. Overview o f a computer model s im u la ti n g the po pu la tio n dynamics of Meloidogyne hapla 31 Daily Loop r CALL TEMP W ith in -D a y Loop CALC TEMP CALC DH CALL SUBS WRITE DATA L Figure 3. Flow c h a r t o f a computer model si m u la ti n g the population dynamics o f Meloidogyne hapla a s s o c i a t e d with Allium cepa. 32 Degree Hours ROOT WEIGHT Temp A . /\ Temg_ Nematode Feeding Function Adjusted ROOT WEIGHT Figure k. Nematode Density Nematode Feeding Sites Flow c h a r t o f Subroutine Onion. 33 tem pfac = -9.4925 + (1.8461*temp) - (0.1115*tem p2 + (0.00265*temp/D/D3/U/U) - (0.00002092*161^) No root growth is considered to occur below 10 C or above 30 C. Nematode density is also used to modify th e estim ate of root growth from what would be expected under optimal edaphic conditions. The adjusted root weight is then passed as an input back to Subroutine Nema. Subroutine Nemfeed The feeding function used in the model was determ ined from the experi­ m ent described on pages 69-101 . A decreased influence of each individual nem atode on plant root weight with increasing population levels was observed in this study. In th e model, the percent root reduction attrib u ted to each nem atode is described by the following equation: 0.0003019 + 0.65623 / feednem * DT (feednem = to ta l no. nemas feeding). The im pact of nem atode feeding on plant root weight is decreased a fte r th e accum ulation of 8000 degree hours, to re fle c t the differential tolerance of new seedlings and established plants to nematode parasitism . Subroutine Nema (Figures 5, 6, 7) Nematodes in the model are divided into seven life stages; eggs, soil juveniles, early second-stage juveniles, la te second-stage juveniles, third/fourthstage juveniles, pre-ovipositing adults, and ovipositing adults. The true life stages of M. hapla were subdivided or combined to more accurately estim ate nem atode development and survival. Early second-stage juveniles, for example, are expected to have th e g re a test m ortality level, since i t is this stage th a t must 3* select and establish a feeding site within host root tissue. In co n trast, third- and fourth-stage juveniles do not feed and develop rapidly. C urrently, rates of nem atode development are computed solely from soil tem perature d ata. The survival of early second-stage juveniles is considered to be 80%, and 100% for the other root-inhabiting stages. These values were obtained from the experi­ m ent described on pages 124-133. In the model, eggs develop into soil juveniles (Figure 5). A developmental subroutine is used to progress soil juveniles to death rath er than to an advanced developm ental stage. Soil juveniles die in 14 days when soil tem perature is less than 20 C, in 7 days when soil tem perature is above 20 C, and in 1000 days when soil tem perature is less than 9 C. The la tte r condition was imposed to approxim ate the delayed development of nem atodes in a sta te of cryptobiosis. All juveniles in the soil are available for root ingress. The number of juveniles sucessfully penetrating roots, however, is a function of several factors. The mobility of juveniles in the soil is g reatest a t 20 C and is reduced to negligible distances when tem peratures are less than 10 C or g reater than 30 C (Wallace, 1963). The carrying capacity of onion root system s is estim ated to be 3000 when plants are less than 34 days old and 1000 when plants are g re a ter than 34 days old. These esitm ates are based on observations and were not confirmed experim entally. The number of juveniles penetrating roots is reduced by 23% in plants with a mycorrhizal association, according to an experim ent described on pages 161-173. Within the root, juveniles progress through th ree developmental stages (Figure 6). stages. Second-stage juveniles are subdivided into early and la te second Early second-stage juveniles are nem atodes th a t have recently pene- 35 Temp Temp Temp Temp Death EGGS SOIL LARVAE Temp O V ER -W EGGS T em p ONION FEMALES MYCO ROOT LARVAE Figure 5* Flow c h a r t o f Subroutine Nema from the egg s t a g e u n t i l the p e n e t r a t i o n of A11iurn cepa ro ot s by Heloldoqyne hapia j uven i 1e s . 36 Temp Temp Temp Temp Temp Temp EARLY L2 LATE L2 L 3 -L 4 w >• a o Figure 6. Flow c h a r t of Subroutine Nema: s ta g e s of Meioidoqyne h a p l a . the r o o t - I n h a b i t i n g 37 tra te d root tissue and have not begun feeding. L ate second-stage juveniles are sexually-differentiated and feeding nem atodes. The third and fourth juvenile stages are combined. Developmental ra te s for the juvenile stages were obtained from th e lite ra tu re (Tyler, 1933; Vrain e t al, 1978; Wong and Mai, 1973a). The minimal ra te of developm ent, considered to occur a t 22 C, is 10 days for early second-stage juveniles, 6 days for la te second-stage juveniles, and 2 days for the combined third- and fourth-stage juveniles. All juveniles successfully m aturing are assumed to become adult fem ales (Figure 7). Pre-ovipositing fem ales are distinguished from ovipositing fem ales, due to the relatively long period of tim e required for reproduction to commence. The minimal ra te of developm ent for pre-ovipositing fem ales is 8 days when soil tem perature is 25 C. Egg production is determ ined by the number of ovipositing fem ales (i.e., is density dependent). An optim al egg output of 14 eggs/fem ale /day is modified according to soil tem p eratu re and nem atode density. Subroutine Soiitemp This subroutine reads and converts bihourly soil tem perature data to an average daily tem perature value. If a tim e step less than one day is used in th e simulation, the average tem perature for each iteratio n is calculated using function FNL. The model may also be executed using a constant tem perature input. Subroutines Model 1. Model 2, Model 3 These subroutines were developed by Dr. R. L. Tummala of Michigan S tate University to model the developm ent of Oulema melanopus. Each stage is 38 Temp Temp Days CO >1 CO Q -----Temp Temp Death t f OVIPOS. FEMALES FEMALES /K i A Nematode Density Nemas Figure 7 Flow c h a r t o f Subroutine Nema: ha pla. a d u l t s ta g e s o f Meloidogyne 39 divided into ten age classes in the subroutines and both in tra - and inter-stage development are calculated. For a further discussion see Tummala e t al. (1974). Model Output The density of eggs, soil-inhabiting nem atodes, and the five nematode stages occurring within plant roots are computed daily. D ata on th e to tal number of feeding sites within root tissue, th e to ta l number of feeding nem atodes, and root weight are also available. The Julian day and accum ulated degree hours (base 9 C) are printed with the above inform ation a t each daily iteration. (The com puter code for th e model is presented in Appendix I.) PROBLEM FORMULATION The study of population dynamics deals with the abundance and distribution of organisms. The abundance of M. hapla is determ ined by th e survival of these nem atodes through tim e, the ra te a t which surviving nem atodes com plete their life cycle, and th e fecundity of fem ale nem atodes. These population attrib u tes, in turn, are influenced by the quality and quantity of food reserves, clim atic conditions, and concom itant soil biota. It is difficult to evaluate the population dynamics of M. hapla in onion production system s without a prelim inary understanding of the biology and ecology of this nem atode. For this reason, only a precursory examination of the population dynamics of M. hapla under agricultural conditions was attem p ted . The m ajority of my e ffo rts were directed toward research needs th a t can be addressed in controlled laboratory studies, but yet are useful for investigations on the population dynamics of M. hapla. The identified needs were as follows: 40 1. Study the response of the onion agroecosystem to M. hapla. determ in­ ing the occurrence, pathogenicity, and persistence of M. hapla associa­ ted with Allium cepa in Michigan. 2. Describe the suitability of A. cepa for th e increase of M. hapla populations. 3. Examine the w ithin-generation dynamics of M. hapla contributing to the increase of this nem atode in Michigan. ft. D eterm ine the influence of other components of the onion agroeco­ system on population a ttrib u tes of M. hapla. SYSTEM IDENTIFICATION AND MENSURATION OF SYSTEM COMPONENTS A subset of all M. hapla in Michigan, those infecting A. cepa. was th e object of concern for this research. M. hapla associated with other crops were not considered in order to minimize variability in m easurem ents of populaion a ttrib u tes. The rationale for this decision was based on observations th a t the host plant is the prim ary facto r influencing the increase of Meloidogyne populations. Oviposition by M. javanica. for example, is alm ost tw ice as great on tom ato as on sugarcane (Balasubramanian and Rangaswami, 1963). For field experim ents, a population of M. hapla was defined to be those nem atodes infecting areas of onion plants th a t could be spatially dem arcated from other areas containing onions. Movement between populations was assumed to be minimal, since only one or two generations of M. hapla normally occur during one growing season in Michigan. Some passive dispersal of nem atodes due to wind, w ater, or machinery may have occurred prior to planting and harvest, since soil-inhabiting juveniles are most prevalent a t these tim es. The passive relocation of M. hapla. however, was assumed to be a random event and of negligible im portance to th e ontogeny of nem atodes during th e experim ental period. The onion agroecosystem consists of many components th a t influence the population dynamics of M. hapla (Figure 1). considered only two components affecting The research discussed here hapla levels; the first order interaction with A. cepa and th e second order interaction with th e VAM Glomus fasciculatus. Abiotic components of the environment which were monitored during experim entation were soil tem perature and m oisture. The association of M. hapla. A. cepa. and G. fasciculatus was characterized in greenhouse experim ents. Population attrib u te s of M. hapla were determ ined using nem atodes infecting individual A. cepa plants. D ata from the greenhouse studies were used to supplement observations from field studies and to provide the basis for further experim entation. One of the major goals of this research was to m easure the influence or pathogenicity or M. hapla to A. cepa. The pathogenicity of any organism to a plant is assessed by measuring the ability of th a t organism to incite disease. Disease, in turn, can be defined as "any disturbance brought about by a pathogen or an environmental factor which in terferes with m anufacture, translocation, or utilization of food, mineral nutrients, and w ater in such a way th a t the affected plant changes in appearance and/or yields less than a normal, healthy plant of th e same variety" (Agrios, (1978). Param eters of plant growth are generally measured to assess the ex ten t of disease exhibited by a plant, since "growth represents the excess of constructive over destructive m etabolism " (Sinnot and Wilson, 1955). Param eters of to tal plant weight and weight of component plant parts were used m ost often in my analyses. Weight m easurem ents were easy to obtain and standardize. In many of the experim ents, particularly those conducted in the greenhouse, the weight of fresh rath er than dry plant m aterial was obtained, because the plants tended to be small and the m ensuration of dry plant m aterial would have required specialized equipment and expertise among employees. Fresh weight is generally not as reliable an indicator of plant growth as dry w eight, since fluctuations in the w ater content of plants can cause changes in weight th a t are not due to the increase of plant tissue. A significant (P=0.05) linear relationship between fresh and dry weights was obtained for my experi­ ments, however, dem onstrating th a t fresh weight was suitable as a t least a relative indication of plant growth (Figure 8). The relationship betw een the fresh and dry weight of the A. cepa grown under field conditions was more variable, but yet still statistically significant (Figure 9). I also measured the size of plant components in some experim ents. Leaf area was assessed by a Li-cor leaf area m eter. The same apparatus was used to m easure root area for one experim ent, but the practice was discontinued because of the difficulty of accurately measuring individual roots. Root length, measured by hand with a ruler, proved to be a more satisfacto ry indicator of the size of the root system . The number of growing tips in a root system was also recorded for some experim ents. The volume of onion bulbs was assessed by measuring the volume of w ater displaced when the bulbs w ere im m ersed. Procedures used for the inoculation and quantification of vesicular— arbuscular mycorrhizal (VAM) fungi w ere adapted from previously published techniques (Schenck, 1982). Fungal chlamydospores were used as inoculum in all U) o £ >- DC O U) Lx. < Y - 0.0633X - 0.0291 U! _l R* - 0.9245 O O. 25 LEAF FRESH WT (g ) to U) DC Y - 0.0552X + 0.0054 Ld R1 - 0.7722 o 5 10 15 20 2 LEAF FRESH WT (g ) Figure 8. R e l a ti o n s h ip between f r e s h and dry l e a f weights of Krummery Special (A) and Downing Yellow Globe (B) A11turn cepa grown in the greenhouse. I/)—. CM Y = 0.0843X R2 = 0.8153 0.0796 o - (g ) CM WT ta- LEAF DRY K o - m ID O. 50 100 150 200 250 LEAF FRESH WT (g ) Figure 9. R e l a ti o n s h ip between f r e s h and dry l e a f weights o f Krummery Special A111um cepa grown under f i e l d c o n d i t i o n s . 45 experim ents for several reasons: (1) it was easy to quantify and standardize the dose received by each plant, (2) spores could by ex tracted from soil samples simultaneously with nem atodes, and (3) th e possibility of contam ination of inoculum by other organisms was minimal. Spore density in th e soil was used to confirm the colonization of roots by VAM and to ch aracterize the development of th e fungal association. The presence of spores is not as reliable an indicator of VAM colonization as is the presence of fungal hyphae, vesicles, and arbuscules in root tissue, but does dem onstrate whether or not colonization has occurred. Enumeration of spores provides a gross measure of the ex ten t of colonization. I also attem p ted , unsuccessfully, to quantify VAM colonization in root tissue. To properly visualize VAM it is necessary to clear roots of all internal structures. U nfortunately, all clearing procedures destroyed th e nem atodes present in root tissue. To minimize the loss of M. hapla within roots, ten 2 cm sections from each root system were stained for VAM, and the rem ainder of th e root system was stained for nem atodes. This amount of root tissue proved to be too little for assessing the ex ten t of fungal colonization in large root system s, and too large in proportion to the size of root systems produced in most of my greenhouse experim ents. In fa c t, the small A. cepa root system s examined in the greenhouse experim ents, particularly in the first six weeks a fte r seeding, were not am enable to mycorrhizal analyses, since both the fungus and nem atode entered roots near the growing root tips. Removing any tissue a t all destroyed a significant proportion of the nem atodes inhabiting th a t root system . Nor was it possible to increase the size of greenhouse experim ents to include plants for mycorrhizal analysis alone, due to th e size of the controlled-tem perature facilities available. The lim itations imposed on experim ental design by assessing 46 VAM only through the quantification of soil spore levels were not serious for the experim ents described here, since the goal of these experim ents was to ascertain what, if any, life-stage or process of M. hapla is influenced by the presence of VAM. Simultaneous quantification of mycorrhizae and nem atode colonization will be essential to the further elucidation of the relationship between these two organisms and plant growth, and should be a high research priority. The accurate assessment of M. hapla population levels was im portant to my research. The aggregate distribution of nem atodes in th e field and the difficulty of extracting nem atodes from the soil medium are constraints common to all nem atological research. To minimize the variability associated with the p attern of nematode distribution, experim ents in the field were conducted in an area not larger, and generally sm aller, than 15.4 x 30.8 m. Microplots were also used, providing the benefits of a small confined a rea and a variable clim atic environment approaching actual field conditions. In all field experim ents a large number of soil cores relative to the sample area were obtained for each soil 3 sample. The samples were thoroughly mixed before a subsample of 100 cm was removed for the assay of nematodes and mycorrhizal spores. Roots were carefully removed from each sample and stained to reveal the nem atodes within. Entire root systems were stained for the observation of nematodes in the greenhouse experim ents, in order to reduce th e sampling error associated w ith pattern s of nem atode distribution within root tissue. Soil from each pot was thoroughly mixed before a subsample was removed for nem atode analysis. The size of the pots was varied in each experim ent, depending on the duration and expected level of nem atodes within the soil. For exam ple, small pots were used when the presence of many second-stage juvenile M. hapla was anticipated. The *•7 duration of most experim ents, however, was not sufficient for the production of a second nem atode generation and levels of M. hapla in th e soil rem ained low. Sampling error associated with those M. hapla life stages inhabiting root tissue was small, since en tire root systems and all root fragm ents in th e soil were examined for nem atodes. In co n trast, th ere was sampling error in enum erating second-stage M. hapla in th e soil. Error in estim ating soil levels of nem atodes is introduced in the (1) collection of samples, (2) storage of samples, and (3) extraction of nem atodes from the soil. The process of extracting nem atodes from soil is variable between nematology facilities and is subject to many sources of error (Kotcon, 1979; M oriarty, 1960; Skellam, 1962). I conducted an experim ent to assess the error and variability associated with extracting M. hapla juveniles from a muck soil (Appendix II). The efficiency of my extraction procedure, a modified centrifugation-flotation technique (Jen­ kins, 1964), was 35% and independent of nem atode density. The d ata presented in this document has not been adjusted by this ex tractio n efficiency facto r and so re fle c t the actual number of nem atodes obtained from th e soil extraction procedure. CHAPTER L Studies on the Occurrence, Pathogenicity, and Increase of Meloidogyne hapla Associated With Allium cepa Meloidogyne hapla is a parasite of many plant species and is an economic­ ally damaging pathogen of a wide range of agricultural crops. It is one of the m ost im portant pests in Michigan and has been the subject of much research. I conducted several studies to clarify th e interaction between M. hapla and Allium cepa. The goals of this research were twofold: (I) to provide inform ation useful to Michigan growers specializing in muck vegetable production and (2) to explore the ecological relationship betw een a nem atode pathogen and its plant host. The specific objectives of the experim ents described here were: (1) to determ ine the incidence of M. hapla associated with A. cepa in Michigan, (2) to assess th e pathogenicity of M. hapla to A. cepa, and (3) to assess the suitability of A. cepa for the increase of M. hapla populations. 48 Survey of Michigan Onion Acreage for Meloidogyne hapla and Other Phytoparasitic Nematodes INTRODUCTION Seventy-eight genera of plant-parasitic nem atodes have been observed from agricultural sites in Michigan (Knobloch and Bird, 1981). Many of these genera are associated with Allium cepa and other vegetables grown in muck soils. B. G. Chitwood, in a 1953 report, listed Ditylenchus spp., Meloidogyne spp., Pratvlenchus spp., Helicotylenchus spp., and Trichodorus spp., as the most im portant pests of A. cepa in Michigan. Since little is known on the suitability of A. cepa as a host to phytoparasitic nem atodes, and in particular to Meloidogyne hapla, I conducted a survey of ca 5% of th e onion acreage in Michigan. Each site in the survey was sampled th ree tim es to d e te c t changes in the population levels of phytoparasitic nem atodes throughout the growing season. MATERIALS AND METHODS Farm s from the following counties were selected for the survey: Ingham, Calhoun, Lapeer, O ttaw a, and Newaygo (Figure 10). Survey p articipants were selected from lists of growers provided by the Michigan Cooperative Extension Service D istrict H orticultural Agents serving these areas. Onion acreage was not a criterion for selection. Consequently, farm s of all sizes with onion acreage ranging from 10 to 150 acres were included in the survey. Each grower provided inform ation on 1) onion cultivar, 2) preceding crop, and 3) pesticides used. Observations on soil type, use of windbreaks, e tc . were recorded when the samples were collected. 50 1c * 4 * i e.\o r u c o \ momi» o * \ a l fcha r*Al**UA\C*AWfO\oscoo* \m iSU U rtd*QlCQM . \O U M A * \i0 iC 9 tA f so h* co Ld Lu I— Q- Ld 100000 0000 INOCULUM DENSITY "3 B K \ Ld OC lu * » _ N. CM »z Y - LOG X/(2.4290HJ0G X - 8.8300) R? - 0.7428 Ol z - 0000 3 Figure 13. ° 100000 INOCULUM DENSITY R el a ti o n s h ip between fr e s h weight o f Krummery Special Allium cepa and Inoculum d e n s it y of Meloidogyne hapla including (A) and excluding (B) and inoculum level o f 30,000 eggs per plant. 77 from the inoculum tre a tm e n t of 30,000 M. hapla per plant, however, the relation betw een inoculum level and mean plant fresh weight a t harvest (Figure 13B) was described by a negative exponential equation: Y - logX / (2.4290 logX - 8.83) where: Y = mean to ta l plant fresh weight in grams X = initial number of M. hapla eggs inoculated/plant Root fresh weight of KS plants inoculated with 15,000 M. hapla eggs was significantly (P=0.05) reduced as compared to plants inoculated with 1000 nem atode eggs or non-inoculated control plants, but there was no difference in root a re a betw een any tre a tm e n t (Tabie 11). Root fresh weight was not different betw een DYG plants inoculated with 100 to 15,000 M. hapla eggs, although root area was significantly (P=0.05) less for plants inoculated with 10,000 eggs as compared to plants receiving 100 eggs (Table 11). An examination of fresh KS root weights over tim e for plants inoculated with 15,000 to 40,000 M. hapla eggs shows th a t root weight was increasingly reduced with increasing inoculum levels, except for th e tre a tm e n t level of 30,000 M. hapla eggs per plant (Figure 14). Similar but more pronounced trends were noted for the relationship betw een mean length of th e KS root system , inoculum level, and tim e (Figure 15). Fresh shoot weight of KS plants inoculated with 15,000 M. hapla eggs was significantly (P=0.05) less than th a t of plants inoculated with 1000 eggs (Table 12). There were no differences in shoot dry weight or area of the KS cultivar for inoculum levels ranging from 100 to 15,000 M. hapla eggs per plant. Similarly, shoot fresh weight, dry weight, or area of the DYG cultivar was not d ifferen t for 78 Table 11. Root w ei g h t and a r e a o f two onion c u l t i v a r s in o c u l a t e d w ith one t o f i v e l e v e l s o f Meloidogyne h ap l a o r noni noculated. Root Cultivar inoculum level Fresh wt (g) Area (cm^) Downing Yellow Globe Check 4.92 a 52.60 ab 100 5.89 a 56.80 a 1000 k.lk a 47.34 ab 5000 5.16 a 44.77 ab 10000 4.12 a 41.12 b 15000 4.39 a 44.41 ab 4.70 a 46.48 a 100 4.17 ab 41.50 a 1000 5.12 a 45.86 a 5000 3.64 ab 36.34 a 10000 3.49 ab 35.02 a 15000 2.68 b 3*».95 a Krummery Special Check Values w it h in a column followed by the same l e t t e r a re not s i g n i f i c a n t l y (P = 0.05) d i f f e r e n t according to Duncan's Multiple Range Te s t. 79 Fi g u r e 14. Fresh r o o t weight o f Krummery S p ec ia l A11lum cepa in o c u l a t e d w it h one o f f i v e l e v e l s o f Meioidogyne hap la o r n o n i n o c u l a t e d . The r e l a t i o n s h i p between root fr e s h weight and accumulated degree hours a t base 9 C was described by the equ ation: where: Y = f r e s h root weight In grams X = accumulated degree hours a f t e r seeding Inocuium den s!ty a b R2 0 0.005126 0.000281 0.9783 15,000 0.002246 0.000312 0.6424 20,000 0.003082 0.000278 0.6816 25,000 0.003231 0.000264 0.9571 30,000 0.001400 0.000331 0.9609 40,000 0.001689 0.000293 0.6337 MEAN FRESH ROOT WT (g ) MEAN FRESH ROOT WT ( g ) * B ? 5 o* o o ? 5 K ©> O MEAN FRESH ROOT WT (g ) ro Figure O \k. MEAN FRESH ROOT WT (g ) MEAN FRESH ROOT WT (g ) + □ ro ro 81 Figure 15. Root length (cm) o f Krummery Special A111urn cepa Inoculated with one of f i v e le v e l s o f Meloldogyne hapla or noninoculated. The r e l a t i o n s h i p between root length and accumulated degree hours a t base 9 C was describ ed by the equation: where: Y « ro o t len g th in cen tim ete rs X = accum ulated degree hours a f t e r seeding Inoculum dens i ty a b R2 0 2.223876 0.000295 0.4443 15,000 1.141120 0.000290 0.7539 20,000 1.295380 0.000279 0.5115 25,000 2.017317 0.000248 0.9224 30,000 1.306399 0.000284 0.9831 40,000 1.035552 0.000272 0.8013 MEAN ROOT LENGTH (c m ) MEAN ROOT LENGTH (c m ) 600 600 0 200 400 0 »_i ^ 200 400 I ■ ■ .t i i ■ 600 600 ea * □ M O MEAN ROOT LENGTH (c m ) 0 200 400 600 800 X Q Figure o 15- MEAN ROOT LENGTH (c m ) 0 200 400 600 MEAN ROOT LENGTH (c m ) 600 0 * B O 200 400 600 600 +B w M O X 'X 83 Table 12. Fresh and dry weights and a r e a s o f leaves of two onion c u l t i v a r s in oc ula ted with one of f i v e le v e l s o f Meloldogyne hap la o r noninoculated. Leaf C u l t i v a r and inoculum level Fresh wt (g) Dry wt (g) 2 Area (cm ) Check 14.45 a 0.77 a 115.65 a 100 16.36 a 0.69 a 128.33 a 1000 13.18 a 0.64 a 99.16 a 5000 15-49 a 0.74 a 137.22 a 10000 12.60 a 0.75 a 106.09 a 15000 14.18 a 0.61 a 118.00 a Check 13.59 ab 0.68 a 102.47 a 100 12.64 ab 0.66 a 99.43 a 1000 14.49 a 0.79 a 107.34 a 5000 12.79 ab 0.68 a 100.62 a 10000 12.25 ab 0.71 a 98.69 a 15000 9.25 b 0.52 a 73-99 a Downing Yellow Globe Krummery Special Values w it h in a column followed by the same l e t t e r a re not s i g n i f i c a n t l y (P = 0.05) d i f f e r e n t according to D u n ca n 's M ulti pl e Range T e s t. 84 inoculum densities of 100 to 15,000 M. hapla per plant. D ifferences in fresh shoot weight with increasing inoculum levels of 15,000 to 40,000 nem atode eggs per plant were not as g reat as the differences in fresh root weight or length (Figure 16). As with root weight and length, an inoculum level of 30,000 eggs per plant had less im pact on plant growth than did inoculum levels of 20,000 or 25.000 eggs per plant. The mean fresh bulb weight and volume of KS onions inoculated with 15.000 M. hapla eggs per plant was significantly (P=0.05) less than th a t of non­ inoculated control plants, but was not different than th a t of plants inoculated with from 100 to 10,000 eggs per plant (Table 13). Plants inoculated with 15,000 to 40,000 nem atode eggs per plant did not form bulbs, due to the length and tim e of the experim ent. There was no difference in th e fresh weight or volume of DYG onions inoculated with 100 to 15,000 eggs per plant. For the experim ent using inoculum densities ranging from 15,000 to 40,000 \1. hapla. levels of eggs d etected in soil samples decreased for all treatm en ts until the fifth sampling d ate (ca 12730 DH), a fte r which tim e an increase was observed (Figure 17). A positive correlation between egg inoculum level and egg density/100 cm soil was noted throughout th e experim ent. Levels of M. hapla juveniles in the soil were g re a test 16 days a fte r planting (ca 4604 DH) for all tre atm en ts and steadily declined th e re a fte r (Figure 17). Again, a positive correlation between egg inoculum level and the density of juveniles was apparent. A significant (P=0.05) linear relationship between inoculum density and the number of juveniles/100 cm soil was observed a t the second sampling date (ca 4604 DH), indicating th a t th e proportion of eggs th a t hatched was independent of inoculum density (Figure 18). 85 Fig ur e 16. Fresh s ho ot weig ht o f Krummery S p eci al A11ium cepa in o c u l a t e d w ith one o f f i v e l e v e l s o f Meloidogyne hapla or noninoculated. The r e l a t i o n s h i p between shoot fr e s h weight and accumulated degree hours a t base 9 was de scr ibe d by the equ ation: where: Y = fr es h shoot weight in grams X = accumulated degree hours a f t e r seeding Inoculum dens 1ty a b R 0 0.030426 0.000213 0.9639 15,000 0.018398 0.000228 0.8746 20,000 0.018088 0.000218 0.6401 25,000 0.024257 0.000199 0.9938 30,000 0.045851 0.000178 0.9246 40,000 0.012702 0.000231 0.7757 MEAN FRESH SHOOT WT (g) MEAN FRESH SHOOT WT (g) o \o MEAN FRESH SHOOT WT (g) ©* oo ON MEAN FRESH SHOOT WT (g) o X o MEAN FRESH SHOOT WT (g) 87 Table 13* Bulb w ei g ht and volume o f two onion c u l t i v a r s I n o c u l a t e d w ith one o f f i v e l e v e l s o f Meloldogyne h a p l a o r nonInoculated. Bulb C u l t i v a r and inoculum level Fresh wt (g) Volume (cm^) Downing Yellow Globe Check 1-45 a 1.81 a 100 1.26 a 1.42 a 1000 1.14 a 1.25 a 5000 1.26 a 1.50 a 10000 1.39 a 1.66 a 15000 1.07 a 1.06 a 1.16 a 1.47 a 100 1.07 ab 1.22 ab 1000 1.10 ab 1.28 ab 5000 0.98 ab 1.19 ab 10000 1.04 ab 1.08 ab 15000 0.68 b 0.80 b Krummery Special Check Values w it h in a column followed by the same l e t t e r are not s i q n i f i c a n t l y (P - 0.05) d i f f e r e n t according to Duncan's M ul ti p le Range T e s t. 88 o D -n CO X 15.000 EGGS 0 20,000 EGGS + 25,000 EGGS 30,000 EGGS ❖ 40,000 EGGS J C/> OO ( /) w DEGREE HOURS (b a s e = 9 C) Figure 17. Levels o f Meloidogyne hapla eggs and j u v e n i l e s In 100 cm^ o f s o i l surrounding Allium cepa Inoculated with one o f f i v e le v e l s o f nematodes o r nonlno cu iated . 89 o to CM Y = 0.006396X - 29.42 O o R“ = .9892 CM o (/) Co o J2 o o o LU o LxJ > ID o to ~ r 10 20 ~~r INOCULUM DENSITY * Figure 18. “ 1 40 30 10* Levels o f Meloidogyne hapla j u v e n i l e s in 100 cm of s o i l 460*1 degree hours a f t e r in o c u la ti o n and p l a n t i n g of A11lum c e p a . 90 There was a significant (P=0.05) linear relationship between the initial inoculum density and th e final observed density of M. hapla per 1.0 g of root tissue for DYG plants inoculated with 100 - 13,000 eggs per plant (Figure 19A). The relation between initial inoculum density and final root levels of nem atodes for the KS cultivar was described by a curvilinear function, since inoculum densities from 1000 - 13,000 eggs resulted in similar final nem atode densities for this cultivar (Figure 19B). The mean number of M. hapla per 0.1 g of root tissue for KS onions inoculated with nem atode egg densities ranging from 13,000 40.000 per plant was g reatest 34 days a fte r planting (ca 4406 DH) and decreased until the experim ent was term inated 73 days (ca 21741 DH) following planting (Figure 20). The density of hapla per 0.1 g of root tissue was not different betw een treatm en ts (inoculum levels) on any sampling d ate. Similarly, the numbers of juvenile and adult M. hapla per root system were not significantly different betw een tre a tm e n ts, although plants inoculated with high levels of eggs tended to support high numbers of nem atodes (Figure 21). The ranking of the tre a tm e n ts in term s of nem atode density/plant, however, was not consistent throughout the experim ent. Plants receiving th e highest inoculum level supported th e lowest nem atode population levels 16 and 30 days a fte r planting. Plants receiving the low est inoculum level did not support the least number of nem atodes on any sampling d ate. In the experim ent based on nem atode inoculum densities of 13,000 to 40.000 eggs per plant, th ere was very little correlation betw een the final density of nem atodes per root system a t 21741 DH a fte r planting and the initial inoculum density, nem atode density 4604 or 9907 DH a fte r planting, or length of the root system 4604, 9907, 14766, or 21741 DH a fte r planting (Table 14). Initial 91 O O cv DOWNING YELLOW GLOBE Y - 0.6881 (LOG X) - 102® R* - 0.8317 o> o 8 e t o ♦ o z z JS 10 100 1000 10000 100000 2 INOCULUM DENSITY o o cv KRUMMERY SPECIAL Od Y - 3.0314(LDG X) - 0.4231 (LOG X*) - 3.3854 R* - 0.8516 O) o 8 1 o « o z z < LlI 10 100 1000 10000 100000 2 INOCULUM DENSITY Figure 19. R e la tio n sh ip between th e inoculum d e n sity o f Meloidogyne hapla eggs and th e number o f nematodes recovered p er gram ro o t tis s u e from Downing Yellow Globe (A) o r Krummery S pecial (B) A111 urn c ep a . 92 o O' o J— O O CxL U) oo O *" \ e & rfi X 15,000 EGGS 0 20,000 EGGS P, = + 25.000 EGGS K 50,000 EGGS ❖ 40,000 EGGS 6 2 z: I < LU t 8 r IS 22 DEGREE HOURS (b a s e 9 C) Figure 20. Number of Meloidogyne hapla in 0.1 gram of AH iurn cepa ro o ts inocula ted with one of f i v e le v e l s o f nematodes o r non in oc ula te d. 93 X 15,000 EGGS o 2 0 ,0 0 0 EGGS □ JO II + SYSTEM o o ■ 25 ,0 0 0 EGGS Q X 3 0 ,0 0 0 EGGS ❖ 4 0 ,0 0 0 EGGS r> < O + S I ft £ ft o ° t —r—i—i—|—i—i—i—i—|—i—i—i—i—|—i—i—i—i—[—i—i—r—i—| 0 5 10 15 20 25 o 0 -1 Csl < !> X + O Ul > o - MEAN NO. M. hapla/ROOT ¥ + * ♦ ❖ a X * 0 8 * X ♦ no. spores Nematode level and crop J u l i a n day P la n t Harvest 140 170 210 238 257 KS A. cepa 25.5 a 38.0 ab 20.0 ab 18.5 a 15.5 a DYG A. cepa 56.5 b 58.5 c 25.5 be 30.5 abc 28.0 a SUS D. c a r o t a 53.5 b 78.0 d 20.0 ab 26.0 abc 19.0 a RES D. c a r o t a 39.5 ab 31.5 a 45.0 d 38.5 be 89.0 be Sudax 50.0 ab 42.5 abe 38.0 cd 27.0 abc 39-5 ab KS A. cepa 32.0 ab 42.0 abc 15.0 a 23.0 ab 15.0 a DYG A. cepa 36.5 ab 31.0 a 17.0 ab 26.0 abc 14.0 a SUS D. c a r o ta 36.0 ab 50.5 be 30.0 be 33-5 abc 56.5 ab RES D. c a r o ta 34.5 ab 30.5 a 29.0 be 44.5 c Sudax 39.0 ab 28.0 a 21.5 ab 29.0 abc Pj = High M. hapla Pj = Low M. hapla Values w i t h i n a column followed by the same l e t t e r a r e not s i g n i f i c a n t l y (P = 0.05) d i f f e r e n t according to Duncan's M ult ip le Range T e s t. 3 KS = Krummery S p eci al / DYG ■ Downing Yellow Globe SUS ■ _M. h ap l a s u s c e p t i b l e / RES - fl. h ap l a r e s i s t a n t 132.0 c 28.0 a 200 P, = LOW M. hapla. P. = HIGH M. hapla FRESH CM I 1 v m KS Figure 2k. 1 P m DYG KS 100 WT 1 NUMBER (k g ) 53 1 DYG Number and f r e s h weight o f Krummery Special (KS) and Downing Yellow Globe (DYG) Allium cepa produced pe r 6.15 m row. e 26. Regression s t a t i s t i c s f o r the r e l a t i o n s h i p between Meloidogyne an d/o r P r aty le nc hus spp. and the weight of A11ium c e p a . (Y)3 (x)b Regression equation KS yield Meloidogyne Y - 1 2 .8 0 - KS yield Pratylenchus Y = KS yield Meloidogyne 2 .7 2 + 9.531X2 R2 P d 0.2640 .19 0.5979 .02* Pratylenchus Y = 4.6 7 - 1.623X1 + 3.729X2 0.7285 .05* DYG yield Meloidogyne Y = 12 .4 4 - l.SOSXj^ 0.1010 .44 DYG yield Pratylenchus Y = 5 .9 6 - 4.864X2 0.1241 .39 DYG yield Meloidogyne Y = 8 .0 3 - 0.434X1 + 1.523X2 0 .1454 Pratyelnchus Total weight (lb) of onions harvested In a 6.15 m row. LoglO(Initial nematode density + 1) / 100 cm C Y = yield d / X^ = Meloidogyne density significant at P = 0.05 level. / ^ soil. = Pratylenchus density 120 resistant carrots damaged by M. hapla. Fifty-five percent of the M. hapla- susceptible cultivar were disfigured, as compared to 24% of th e resistan t cultivar. In the area with low initial M. hapla density th e M. hapla-resistant cultivar yielded b e tte r than th e susceptible cultivar, but both cultivars produced similar numbers of galled carrots (Table 27). Yields of the resistant D. caro ta cultivar were significantly (P=0.05) reduced in the area infested with M. hapla, whereas similar yields of th e susceptible cultivar were harvested from each area. The proportion of suscepti­ ble carrots which were disfigured and galled, however, was much reduced in th e a re a with low initial M. hapla levels. DISCUSSION This experim ent was designed to evaluate the growth of five crops and M. hapla populations without the use of pesticides or th e inoculation of nem atodes. Initial densities of M. hapla were discrepant between the two areas used for this experim ent, but were not different enough to produce results comparable to te sts where nem atode levels were regulated by inoculation (see previous section). The trends noted in this experim ent do deserve atten tio n , however, since the experim ental design approximated natural conditions b e tte r than could be achieved by the artificial manipulation of th e field environm ent. The sim ilarity in yield between cultivars and the absence of a significant linear relationship betw een initial M. hapla density and onion yield may have been due to the low numbers of nem atodes present in th e experim ental site. Previous studies in the greenhouse showed th a t neither th e KS or DYG A. cepa cultivars are affected by M. hapla densities of 100 eggs per plant. It is possible Ta b l e 2 7 . Number and f r e s h w e ig h t (kg) o f two c a r r o t c u l t i v a r s p e r 6 . 1 5 meter row. Stubby and forked w ith g a l l s Hea1thy Nematode level and crop No. Wt. No. Wt. Stubby and forked wi t h o u t gal Is No. Total No. Wt. Wt. P. - High M. hapla SUS D. c a r o t a 33-0 a 3.48 a 48.8 a 4.20 a 7.75 a 0.73 a 89.5 a 8.55 a RES 0. c a r o t a 66.0 a 6.48 a 23.0 b 1 .70 b 7-75 a 0.58 a 96 .8 a 8.40 a SUS D. c a r o t a 54.5 a 6.45 a 22.5 b 1.95 b 14.00 a 0.90 a 91 .0 a 9-30 a RES D. c a r o t a 103-3 b 11.48 b 24.5 b 1.83 b 15.00 a 1.08 a 175-8 b 14.38 b P. = Low M. hapla Va lu e s w i t h i n a column f o l l o w e d by t h e same l e t t e r a r e n o t s i g n i f i c a n t l y t o Duncan's M u l t i p l e Range T e s t . SUS = M_. h a p l a s u s c e p t i b l e / RES = M_. h a p l a r e s i s t a n t (P = 0.05) d i f f e r e n t a c c o r d i n g 122 th a t densities of M. hapla were not much g reater than this in the experim ental a re a, or th a t the distribution of nem atodes was such th a t relatively few plants were subjected to densities g reater than 100. The two cultivars did respond differentially to M. hapla (Table 26), supporting my earlier findings th a t the KS cultivar is less tolerant of M. hapla infection than the DYG cultivar. Total yields of the M. hapla-susceptible D. caro ta cultivar were compar­ able in the areas with low and high M. hapla levels, but more than tw ice as many nematode-damaged carrots were produced in the area with high M. hapla levels (Table 27). In contrast, carro t yields of the M. hapla-resistant cultivar were significantly (P=0.05) reduced in the area with high initial M. hapla density, despite the fa c t th a t relatively few nem atodes were observed within soil or root samples from this crop (Tables 15, 16). This result suggests th a t despite the reduced ability of M. hapla to infect or reproduce on this cultivar, those nem atodes which do invade roots a ffe c t plant growth more severely than do nem atodes infecting the M. hapla-susceptible cultivar. The reduced number of c arro ts produced by the M. hapla-resistan t cultivar in the area with high initial M. hapla density supports the possibility th a t young carrots of this cultivar can be killed by lower nem atode densities than the suceptible cultivar. Like the M. hapla-resistan t D. caro ta. many crops differ in their ablilty to support nematode infection and to sustain nem atode injury, justifying th e distinction between th e susceptibility/resistance and the tolerance/intolerance of a plant to nematode parasitism (Nusbaum and Barker, 1971). .M. hapla levels decreased on A. cepa and increased on D. carota. confirming the contention of some authors th a t A. cepa is not as good a host for M. hapla as other vegetables commonly grown in muck soil (Bird, 1981; Kotcon, 123 1979; Van Arkel, 1982). The reason for this is not clear, since final densities of M. hapla were not significantly related to the density of VAM spores, Pratylenchus spp., or predaceous nem atodes. The significant relationship between these facto rs and M. hapla density on carro t suggests th a t the failure of M. hapla populations to increase on onion may have been due solely to a ttrib u te s, or lack of a ttrib u te s, of this host plant. D espite the fa c t th e some juvenile nem atodes entered roots of A. cepa. few if any m atured to th e adult stage (Table 16). It is possible th a t parasitism by Pratylenchus spp. was sufficient to reduce the suitability of A. cepa as a substrate for M. hapla developm ent, particularly in light of the significant relationship bew teen th e yield, and presumably the growth, of the KS cultivar and Pratylenchus density (Table 26). A differential number of VAM spores was d e tected in soil samples from A. cepa and D. caro ta (Table 25). Significantly (P=0.05) more spores were associated with the M. hapla-resistan t D. caro ta cultivar. The reason for this is not clear, but may have been a facto r contributing to the reduced number of nem atodes supported by this cultivar (See C hapter II). Sudax did not promote the increase of VAM, which is surprising since sorghum is often used to culture VAM in the greenhouse. Stage-specific Survival of Meloidogyne hapla Associated with Allium cepa. INTRODUCTION E stim ates of the survival ra te s of M. hapla can be useful in understanding and predicting the population dynamics of this nem atode. The increase of a M. hapla population is determ ined by: (1) th e number o f nem atodes recruited into the population by birth or im m igration, (2) the ra te a t which nem atodes develop and reproduce, and (3) the number of nem atodes lost due to m ortality or em igration. The decline of nem atode populations from th e beginning to the end of a single season, or over multiple seasons, has been addressed in several studies (for further discussion see Norton, 1978), but th e d ata obtained provides only a gross estim ate of change occurring within a population and does little to elucidate the biology of the nem atodes studied. The expected survival of each life stage present in populations of M. hapla. or other plant-parasitic nematodes, is not known. For some stages, survival ra te s can be estim ated from data collected for different purposes. For exam ple, exam ination of d ata from studies by Dropkin (1963), Griffin and Elgin (1977), Wong and Mai (1973b), and this author show th a t ca 20 - 38% of M. hapla eggs successfully advance to rootinhabiting stages. In contrast, survival ra te s for th e four life stages of M. hapla inhabiting root tissue are not available. The major objective of this study was to determ ine the survival ra te s for those stages of M. hapla inhabiting A. cepa roots. Field census d ata of M. hapla from the M.S.U. Muck Research Farm was collected during th e summer of 1981 to accomplish this objective. In addition, th e pathogenicity of M. hapla to A. 124 125 cepa was assessed by examining the relationship between nematode density and onion yield. MATERIALS AND METHODS A. cepa (cv Krummery Special) was planted in 3-row beds in range C-17 of the M.S.U. Muck Research Farm on 6/1/81. A fter planting, nine consecutive beds of onions were divided into five 30.77 cm sections. The sections were marked by stakes and numbered from 1 - 43. A soil sample consisting of ca 130 3 cm of soil from a depth of 1 - 15 cm was removed from each section six tim es from planting until harvest: 6/30/81, 7/14/81, 8/12/81, 8/25/81, and 9/22/81. On the last sampling date two soil samples were removed from each section; one from a depth of 1 - 15 cm and another from a depth of 15 - 30 cm . A 100 cm^ subsample was removed from each sample for nematode analysis. The root fragm ents contained in the subsample were removed, weighed, stained in a solution of lactophenol with 0.01% acid fuchsin, and examined for nem atodes using a dissecting microscope. Nematodes w ere ex tracted from th e soil of each subsample by a modified sugar flotation-centrifugation technique (Jenkins, 1964). The onions were harvested on 9/22/81 and the fresh weights recorded. N ematodes within roots were enum erated according to the following scheme: (1) early second-stage juveniles (slender body shape), (2) interm ediate second-stage juveniles (broadened body shape), (3) late second-stage juveniles (broadened body shape with rounded ta il term inus), (4) third/fourth-stage juven­ iles (as late second-stage juveniles with m ultiple cuticles present), (5) pre­ ovipositing fem ales (globose body shape), and (6) ovipositing fem ales (globose body shape with eggs visible). This scheme was selected because of the ease 126 with which these stages could be distinguished morphologically, and because biologically, each stage represents phases in th e life cycle of M. hapla th a t might be expected to have unique ra te s of survival. The la tte r point is im portant, since m ost methods for calculating stage-specific survival assume th a t m ortality ra te s are constant within individual stages. The m ortality of each M. hapla life stage was estim ated using a method developed by Southwood (1966). The counts for each stage were plotted against tim e. The area under the resulting stage-frequency curves was integrated to give the to tal incidence of each stage. Since nem atodes required more than one day to com plete any stage, the to ta l incidence was divided by the tim e required to com plete developm ent of th a t stage. The values obtained were estim ates of the to ta l number of M. hapla entering each life stage. The daily survival for each stage was computed by comparing the to ta l number of nem atodes th a t entered each stage: Sjj = No. in stage II + 1 (S^ = daily survival of Stage II) No. in stage II Developmental tim es for 20 C, the average soil tem perature from the sample site a t a 15 cm depth, were estim ated from the data presented by Tyler (1933), Vrain e t al., (1978), and Wong and Mai (1973a). Separate estim ates of developm ental tim es for the early second-, interm ediate second-, and late second-stages could not be obtained from these studies, so the data for the stages were combined. The estim ated developm ental tim es were: 16 days for the second juvenile stage, 2 days for the combined third and fourth stages, and 10 days from the m olt to adulthood until oviposition. 127 RESULTS Root levels of M. hapla were maximal on 7/29/81 (Julian day 210) and very low a t harvest on 9/22/81 (Julian day 265). On 7/29/81 th ere were an average of 3.40 nem atodes in the root fragm ents contained within 100 cm of soil, or ca 4.277 nem atodes per gram of root tissue. M. hapla levels in the soil decreased from planting until the first sampling date on 6/30/81 (Julian day 181), and then increased until the last sampling a t harvest (Figure 25). Both root and soil nem atode levels were lower a t a depth of 15 - 20 cm than a t a depth of 1 - 15 cm on 9/22/81. Comparing final soil + root densities (P^) to initial soil densities of M. hapla (P.) yields a seasonal ra te of increase of 4.904 nem atodes. All root-inhabiting stages of M. hapla occurred simultaneously on and a fte r Julian day 210 (7/39/81) (Table 28). Levels of second-stage juveniles peaked a t th e second sampling date on Julian day 195 (Figure 26). Third/fourth stage juveniles and pre-ovipositing adults reached maximal levels on Julian day 210. The occurrence of ovipositing fem ales was first noted on the same sampling date, and was g re a test on Julian day 224, 73 days a fte r planting. The survival ra te s for the second, and the combined third and fourth stages were 0.7944 and 1.0763, respectively. The survival of pre-ovipositing adults could not be estim ated since the length of th e ovipositional period was not known. There was a slight yet significant (P=0.05) linear relationship between the peak M. hapla density a t 7/29/81 and the final fresh weight of onion (Figure 27) The mean fresh weight of onions in each 30 cm bed (3 rows) was 1090.92 ± 41.550 g. 128 Ld CD Soil Q* CD Roots ID o 150 200 250 300 JULIAN DAY Figure 25. Levels of Meloidogyne hapla in 100 cm^ s o i l samples c o l l e c t e d from the M.S.U. Muck Research Farm in 1981. 129 Table 28. Incidence o f Meloldogyne h a p l a In 1981 by l i f e - s t a g e . SECOND-STAGE JUVENILES: Date Mean Variance 181 195 210 224 237 1.1333 l.lkkk 1.2889 0.5111 0.9556 3.2711 7-3847 2.1165 0.6943 1.9995 Standard Error 0.2696 0.4051 0.2169 0.1242 0.2981 Average st andard e r r o r o f mean - 23* THIRD/FOURTH-STAGE JUVENILES: Date Mean Variance 195 210 Ilk 237 0.0444 0.4000 0.1778 0.0667 0.0425 0.6844 0.2351 0.0622 Standard Er ror 0.0307 0.1233 0.0723 0.0372 Average standard e r r o r o f mean = kS% PRE-OVIPOSITING FEMALES: Date Mean Variance 195 210 Ilk 237 0.0222 1.5778 1.2000 0.7556 0.0217 4.8217 2.6930 1.4625 Standard Error 0.0220 0.3273 0.2446 0.1803 Average standard e r r o r of mean = 41* OVIPOSITING FEMALES ; Date Mean Variance 210 22^4 237 0.1333 0.9333 0.6889 0.1156 2.5500 1.1810 Standard Error 0.0507 0.2381 0.1620 Average standard e r r o r of mean = 29* 130 □ Second-stage juveniles # Thlrd/fourth-stage juveniles * Pre-ovlpoelttng adult females ❖ Ovipositing adult females O O 150 200 250 JULIAN DAY Figure 26. S t a g e - s p e c i f i c d e n s it y of Meloldogyne hapla i n h a b it in g roo ts o f A1 Hum cepa in 100 cm o f soi 1 from the M.S.U. Muck Research Farm in 19 d l . 131 K O — m- m— o U) o 1/3 Y = 1195 - 23.24X 0 5 10 (R1 = 0.1247) 15 20 M . /tapia/100 cm3 SOIL SAMPLE Figure 27. R el a ti o n s h ip between Meloidogyne hapla d e n s i t y and the fr e s h weight of bulbs o f A11ium cepa ha rv e ste d per 30 cm row. 132 DISCUSSION Eighty percent of the second-stage M. hapla juveniles inhabiting root tissue survived to become third- and fourth-stage juveniles. Survival of the third- and fourth-stage juveniles was ca 100%. These results concur with knowledge on the biology of M. hapla. Second-stage juveniles must locate and establish feeding sites within the root, a process th a t can be influenced by th e number of nem atodes already inhabiting th a t root, and by the nutrient status of th e host plant. It is not likely th a t all nem atodes entering a root will be successful in procurring enough space and nutrients to com plete developm ent. Third- and fourth-stage juveniles, on the other hand, do not feed and are probably not vulnerable to m ortality factors unless th e host plant dies. Second-stage juveniles were subdivided into early, interm ediate, and la te substages for this study because I fe lt it likely th a t survival differed from th e beginning to the end of this stage. U nfortunately, the data could not be analyzed according to this classification, since estim ates of developm ental tim e for each substage were not available. Southwood's method for calculating age-specific m ortality assumes th a t m ortality is distributed equally throughout the stage. If m ortality is heavy a t the onset of th e stage, as may be th e case with M. hapla. then this method will under-estim ate the to ta l incidence of th e stage, and consequently, over-estim ate the m ortality of the stage. If I am c o rre ct in assuming th a t m ost m ortality occurs in the early portion of the second juvenile stage, then the actual survival of second-stage juveniles may be g reater than 80%. There was a four-fold increase in levels of M. hapla from the beginning until the end of this experim ent. The d ata indicate th a t only one generation of nem atodes occurred (Figure 26). Survival of pre-ovipositing fem ales was not 133 calculated. Inspection of the d ata in Figure 26, however, shows th a t it is unlikely th a t all fem ales commenced oviposition before the senescence of onion roots a t harvest. The phenology of A. cepa, then, ex erts th e same pressure on the population increase of M. hapla as if m ortality in an earlier stage had occurred; in either case, the affected nem atode does not replace itself in the population. The early senescence of onion roots may be the major factor lim iting the increase of M. hapla populations relative to those crops in which root growth continues until and beyond harvest. M. hapla was not highly pathogenic to A. cepa in this study, although onion yields were significantly (P=0.05) reduced due to this nem atode (Figure 27). The increase and detrim ental influence of M. hapla associated with A. cepa indicate th a t this crop, or a t least the cultivar Krummery Special, may be a b e tte r host for M. hapla than is generally assumed. GENERAL DISCUSSION The preceding studies dem onstrated th a t M. hapla is present in much of the onion acreage in Michigan and th a t it may be d etrim ental to onion production. M. hapla was pathogenic to A. cepa under greenhouse conditions, and decreased onion yield in a small field plot (Figure 27). Since the numbers of nem atodes necessary to reduce plant growth are not commonplace or uniformly distributed in com m ercial onion production sites, however, this nem atode does not seem to be of major economic significance to A. cepa. Differences in th e reactions of d ifferen t A. cepa cultivars to M. hapla infection were observed, as was dem onstrated in earlier experim ents (Franklin, 1959; Kotcon, 1979; Sasser, 1954) D ifferent cultivars of many plant species vary in their response to nematodes. Generalizations on the nonhost status of a plant species, as have been made for A. cepa, can be misleading and should be avoided. In general, it seems th a t A. cepa is not well-suited for the increase of M. populations, particularly as compared to D. c a ro ta . However, M. hapla does reproduce on A. cepa and populations can increase from the beginning to th e end of a growing season, as evidenced by my greenhouse experim ents and the field census data collected to com pute M. hapla survival rates. The ability of A. cepa to m aintain M. hapla populations should be considered when a muck vegetable rotation is followed. The final levels of nem atode present a t the harvest of A. cepa may not need to be very g re a t to th reaten the production of future crops, especially carro t or celery (Slinger, 1976). Although an onion-carrot rotation is preferable to the monoculture of carro t, A. cepa should not be viewed as a panacea to problems caused by M. hapla. There was no significant relationship betw een nem atode densities of 100015,000 eggs per plant and the increase of M. hapla on the A. cepa cultivar Krummery Special (Figures 19B). For th e Downing Yellow Globe cultivar, however, a significant relationship between inoculum levels of 500 - 15,000 eggs and final nem atode density was observed (Figure 19A). It may be th a t the first juveniles entering KS onions sufficiently a lte r root growth to reduce the number of sites available for the entry of additional nem atodes. By the tim e a flush of new root growth occurs, juveniles in th e soil may have depleted their food reserves and be less able to locate and p en etrate th e new root growth. If so, an A. cepa cultivar more tolerant to M. hapla infection, such as DYG, may have been a more suitable cultivar than KS to te st the density-dependence of M. hapla increase. It is also possible th a t th e numbers of M. hapla entering KS roots 135 increases with increasing inocuium levels, but th a t the survival of nem atodes is reduced when root nem atode levels are high. A ppropriate d ata to te s t this possibility was collected in the greenhouse experim ent using high nem atode inoculum levels. An analysis of stage-specific survival ra te s was not perform ed, however, since the stage frequency counts increased steadily throughout the experim ent and a reasonable end point to each stage could not be estim ated. CHAPTERn Studies on the Interaction Between Meloidogyne hapla and the Vesicular-Arbuscular Mycorrhizal Fungus, Glomus fasciculatus, Associated with Aliium cepa Colonization of plants by vesicular-arbuscular mycorrhizae (VAM) may have a depressive e ffe c t on the increase of phytoparasitic nematode populations. The mechanisms responsible for this influence cannot be identified until the relationship between VAM and nematodes is understood in g reater detail. The major objective of the research presented here was to identify which portion of the nematode life cycle is most affected by VAM colonization in the host plant. Five experim ents were perform ed to: (1) establish whether VAM can limit the increase of nem atodes, (2) identify the influence of mycorrhizal colonization on the location and penetration of plant roots by nem atodes in the soil, and (3) characterize and compare the development and reproduction of nematodes within mycorrhizal and non-mycorrhizal roots. Although the experim ents were conducted with the nematode Meloidogyne hapla. the VAM Glomus fasciculatus. and the host plant Allium cepa. they were intended to provide information th a t might be useful in characterizing other nematode-VAM-piant associations. 136 Influence of the Vesicular-Arbuscular Mycorrhizal Fungus, Glomus fasciculatus. on the Pathogenicity and Increase of Meloidogyne hapla Infecting Allium cepa INTRODUCTION Vesicular-arbuscular m ycorrhizae (VAM) and phytoparasitic nem atodes are both commonly associated with many plant species. The stim ulation and inhibition of plant growth by VAM and nem atodes, respectively, has been described in detail (Safir, 1980; Seinhorst, 1965). The concom itant influence of VAM and nem atodes on plant growth has also been examined but is not yet thoroughly understood. According to some studies, nem atode parasitism is not as deleterious to plants with an established VAM association as to plants inhabited by nem atodes alone. For exam ple, Kellam and Schenck (1980) dem onstrated th a t yields of soybean inoculated with both Meloidogyne incognita and Glomus m acrocarpus were significantly g re a ter than plants inoculated with the nem atode alone. Similarly, Atilano e t al. (1981) found th a t dry shoot weight of grape infected with M. arenaria was increased when plants w ere colonized by G. fasciculatus. It is not clear if the ability of some plants to b e tte r withstand nematode infection when colonized by VAM is due to th e reduction of nem atode population levels, or to the increased tolerance of mycorrhizal plants for nematode parasitism . VAM colonization decreased population levels of H eterodera solanacearum , M. hapla. and M. incognita infecting tobacco, c arro t, and tom ato, respectively (Fox and Spasoff, 1972; Sikora and Schonbeck, 1975; Kellam and Schenck, 1980), indicating th a t VAM adversely a ffe c ts the ontogeny of some 137 138 phytoparasitic nem atodes. Conversely, mycorrhizal plants supported g reater numbers of M. incognita and were larger than non-m ycorrhizal plants (Atilano e t al., 1981), suggesting th a t th e increased plant nutrition associated with VAM colonization enhanced the ability of plants to to le ra te nem atode infection. The interaction between M. hapla and the growth of A. cepa was examined in experim ents described previously (see C hapter I). Correspondingly, the influence of VAM on the growth of A. cepa has been reviewed by Carling and Brown (1982) and Gerdemann (1975). The objective of my research was to assess the im pact of VAM on the relationship betw een JM. hapla and A. cepa. Two experim ents were perform ed to determ ine if: (1) VAM enhances plant growth and has no im pact on M. hapla. or (2) VAM has a positive influence on A. cepa and a negative im pact on M. hapla. or (3) both A. cepa and M. hapla are benefited by VAM colonization. MATERIALS AND METHODS Greenhouse experim ent. A Houghton muck soil with a pH of 6.6 and a phosphorus (P) level of 60 ppm was obtained from th e M.S.U. Muck Research 3 Farm for this experim ent. Approximately 1500 cm o f pasteurized soil was thoroughly mixed with inoculum prior to placem ent into each of 120 clay pots. Inoculation treatm en ts, adm inistered in 5 ml of w ater, were: (1) 10,000 M. hapla eggs, (2) 2000 G. fasciculatus spores, (3) 10,000 M. hapla eggs and 2000 G. fasciculatus spores, or (4) w ater passed over eggs and spores (control). Presum ­ ably, the w ater filtered over the nem atode and mycorrhizae inoculum contained any microbial organisms present in the cultures. One A. cepa seed (cv Krummery Special), pregerm inated for 48 hours, was planted into the middle of 139 each pot. The plants were maintained in the greenhouse for eight weeks and w atered daily. Six replicates of each tre atm en t were harvested 2, 4, 6, and 8 weeks a fte r planting. At each harvest, root, bulb, and leaf fresh weights, and bulb and leaf dry weights were determ ined. Soil samples from each pot (100 cm ) were assayed for nem atodes and fungal spores by a sugar flotation-(1.37 specific gravity) centrifugation technique (Jenkins, 1964). The entire root system of each plant was stained in a solution of lactophenol and 0.01% acid fuchsin. The colonization of roots by VAM was confirmed, and th e nem atodes contained within each root system were enum erated using a stereom icroscope. Plants th a t died during the experim ent were included in the analyses of plant growth param eters but were not considered in the analyses of nem atode population growth, since M. hapla is an obligate parasite th a t cannot develop in the absence of a host plant. A prelim inary experim ent using the same methodology but with a duration of seven weeks was perform ed six months earlier. Microplot experim ent. A. cepa (cv Krummery Special) plants for this experim ent were seeded in the greenhouse and transplanted five weeks later to the M.S.U. Muck Research Farm . Five onion seeds were planted into each of 280 plastic pots containing 217 g (dry wt) of a 1:7 mix of a muck and sand soil with a pH of 6.5 and P level of ca 2 ppm (Bray's P -l extractable). At planting, one third of the pots were inoculated with 2000 G. fasciculatus spores in 5 ml o f w ater. Another third were fertilized with 5 ml of a 8.60 mg/ml solution of KH2PO^ in w ater, adding ca 48 ppm P to stim ulate the growth response obtained using VAM. The final third of th e pots were non-inoculated and received no P fertilization. Pots not inoculated with G. fasciculatus received 5 ml of w ater passed over 140 spores so th a t any microbial organisms inhabiting the VAM culture were present in all three treatm en ts. The nutrient levels in all pots were adjusted using KNO^ (non-P-treated pots) and NH^NO^ (P -treated pots) so th a t only P levels were different betw een tre a tm e n ts. All pots were thinned to one seedling a fte r one week. The tre a tm e n ts were repeated a t transplanting, except th a t 50 cm 3 of soil from a greenhouse culture of G. fasciculatus containing ca 1200 spores and 14 g of superphosphate replaced the original inoculum preparations. In June, ten plants from the same tre a tm e n t w ere planted in a circular p attern into eight aluminum microplots ca 40 cm in diam eter. In addition, four microplots were each planted with eight G. fasciculatus-trea te d plants and fertilized with superphosphate. One half of the microplots containing control, Ptreated , or VAM-inocuIated plants, and all of the m icroplots containing VAM + P -tre ate d plants were inoculated with 2000 M. hapla second-stage juveniles. The nem atode inoculum was delivered in 5 ml of w ater around the roots of each plant. Four replications of the resulting seven treatm en ts were arranged in a randomized block design. The microplots were fum igated with Vorlex (143 1/ha) in May o f the previous year and contained no phytoparasitic nem atodes a t the tim e of transplanting. One plant was removed from each microplot every two weeks for 12 weeks. The plant selected for each destructive sample was predeterm ined by its position in the m icroplot, in order to elim inate bias in the selection of a single plant to represent each m icroplot. Bulb and leaf fresh and dry w eights were determ ined. 3 One gram of roots and 100 cm of soil surrounding each plant were assayed for M. hapla and G. fasciculatus as described previously. All plants remaining a t the final harvest were removed and included in the last data analysis. 141 The change in M. hapla density over th e growing season was com puted by 3 comparing the final and initial density of nem atodes in a 100 cm soil sample: hapla = density (Pf) / initial density (Pj) RESULTS Greenhouse experim ent. Plants inoculated with G. fasciculatus alone were significantly (P=0.05) larger a fte r eight weeks than control plants or plants inoculated with M. hapla (Figure 28). Plants infected with M. hapla were consistently sm aller than non-infected plants throughout th e experim ent, regard­ less of the presence of VAM. By th e end of the experim ent, ten weeks a fte r planting, leaf fresh weights of plants inoculated with th e VAM alone w ere more than tw ice as g re a t as control plants and over ten tim es larger than plants inoculated with M. hapla alone or with M. hapla and G. fasciculatus. differences are also reflected in a comparison of leaf dry weights. These The fresh bulb weight of mycorrhizal plants was also g re a te r than non-m ycorrhizal plants when the experim ent was term inated. The root system s of VAM-treated plants were significantly larger a fte r ten weeks than those of plants inoculated with nem atodes, but were not differen t from non-inoculated control plants. Two, four, and six weeks a fte r planting, th e re was no difference in the levels of M. hapla associated with eith er m ycorrhizal or non-mycorrhizal plants (Figure 29). A fter the first appearance of ovipositing fem ales eight weeks a fte r planting, however, M. hapla density per root system increased both in the presence and absence of G. fasciculatus. To te s t w hether th e increase in nem atode numbers was due to an influx of second generation nem atodes, an analysis of variance showed th a t the density (lo g ^ ) of juveniles during this tim e 142 Figure 28. Mean f r e s h weight (+ st andard e r r o r ) o f A11iurn cepa in oc ul a te d with Meloidogyne h a p l a , Glomus f a s c i c u l a t u s , Meloidogyne hapla and Glomus f a s c i c u l a t u s , and non­ in oc ul a te d c o n t r o l . MEAN LEAF WT (g ) (£> C n 20 o <0 X + G □ NJ CO • MEAN ROOT WT (g ) m m o. I/) MEAN PLANT WT (g ) o 5 10 IS 20 2! (9 X + 0 0 ro O-1 MEAN BULB WT (g ) 0 . 1 » » » 2 3 « 1 ■ ■ ■ i I ■ » ■ » I u 4 S i i 1 ■ ■ ■ ■ I X + 0 Q o X + 0 Q 144 I— 5 g A Meloidogyne hapla 2 s* > f-4 1 A i— m — ' ^ f o ... O Z Z < LJ oviposmoN _ 4r ----- '--- 1--- '--- 1----r—T— '--- 1 1 I 0 2 4 6 8 10 WEEKS 5 g 3 1 1 B Meloidogyne hapla + Glomus fasciculatus x * 5'!! ^ 9 4 O z oviposmoN < "1— 1— i— '— i— 1 i f Ul o 2 4 6 ■— i— «— i 8 to WEEKS Figure 29. Mean number of Meloidogyne hapla (+ st an dar d e r r o r ) In­ h a b i t i n g root systems o f A111 urn cepa inocu la ted with Meloidogyne hapla alone (a ) o r with MeloWoqyne hapla and Glomus f a s c i c u l a t u s (B). 145 was unchanged in the plants inoculated with M. hapla alone, and decreased significantly (P=0.05) in the plants inoculated with both nem atodes and mycorrhizae (Tabie 29). There was no difference between the number of M. hapla observed within mycorrhizal and non-mycorrhizal plants during this experim ent. There was evidence, however, th a t differences in the ra te of nem atode development occurred in the presence and absence of VAM. Six weeks a fte r planting, a t the tim e ovipositing fem ales were first observed, tw ice as many nem atodes (P=0.08) had m atured to the adult stage on plants inoculated with th e nem atode alone (Figure 30). Levels of M. hapla juveniles in the soil were not different between treatm en ts except for week two, when significantly (P=0.05) more nem atodes were observed in pots containing M. hapla with no VAM (Figure 31). Levels of VAM spores were not d ifferen t in pots inoculated with spores alone or with spores and nem atodes until ten weeks a fte r planting (Figure 32). Spore densities associated with both treatm en ts decreased six weeks a fte r planting and then increased by the next sampling d ate. From week eight until week ten, the density of spores in th e soil surrounding nem atode-infected plants was unchanged, but decreased in the soil surrounding nem atode-free plants. Microplot experim ent. There was no difference in the fresh bulb or shoot weights of control, VAM-inoculated, or P-fertilized plants, although control plants tended to be the sm allest of th e three treatm en ts (Figures 33, 3*0. M. hapla infection was not detrim ental to control or treated plants. Similar trends in plant growth were reflected in bulb and leaf dry weights. Significantly (P=0.05) more nem atodes entered and were maintained in plants inoculated with VAM alone than in plants both inoculated with VAM and 146 Table 29. Density of second-sta ge Meloidogyne hapla (logjp) in All 1urn cepa inocu lated with nematodes alone o r with nematodes and Glomus f a s c i c u l a t u s s i x and e i g h t weeks following inocuation. Treatment Mean Standard deviation Ta Pb Meloidogyne hapla 1.6740 Week 6 1.6200 0.246 Week 8 1.1000 0.229 © Week 8 0.235 0.608 CM 1.3240 0.2836 2.879 0.0450 1 Week 6 Meloidogyne hapla + Glomus f a s c i c u l a t u s Value obtained from a two sample T t e s t . b P r o b a b i l i t y t h a t the mean values a r e d i f f e r e n t . 147 o o Meloidogyne hapla Z Meloidogyne hapla + Glomus fasciculatus 3 OL Lu •. _l o < UJ O z < Z lo O " P° or o DL O O' a. a o 4 6 WEEKS Figure 30. Pr oport ion of the t o t a l number o f Meloidogyne hapla reaching the a d u l t s ta g e in Allium cepa in oc ul a te d with Meloidogyne hapla alone o r with Meloidogyne hapla and Glomus f a s c i c u l a t u s (mean values + CI q q j ) . 148 U3 Csl —i o too M ***l °_ • o o o 0 litloidogyns hapla X Meloidogyne hapla + Glomus fasciculatus 1/3■ CO U i o o \ \ \ \ (( \ \ o z < 1/3 • W \\ Id 2 'C N 1------'------1------ '------ 1------ '----- T----- -- ----- 1 2 4 6 8 10 WEEKS Figure 31. Numfaer-of Meloidogyne hapla sec ond-stage J u v e n i l e s In 100 cm s o i l surrounding Allium cepa in oc ula ted with Meloidogyne hapla alone o r with Meloidogyne hapla and Glomus f a s c i c u l a t u s . 1^9 o o Q Control Cvl © JfcloicEojrVtt* taplo+ Glomus fasciculatus X if. /tapta + G. fasciculatus O o CO s \ \ \ N ' CO Ld a: O Q- o (/) ® • \ \ \ ' \ \ \ \ // y y y y ' j: V o z I < $■ Ld ^ V T 4 » -r r l T 6 8 10 WEEKS Figure 32. Mean number (+ s ta nd a rd e r r o r ) o f Glomus f a s c i c u l a t u s in 100 cm s o i l surrounding A11iurn cepa inoc ulated with Meloidogyne hapla alone o r with Meloidogyne hapla and Glomus f a s c i c u l a t u s . VI o Fig ur e 33* Fresh bulb weight o f Allium cepa supplemented wi th phosphorus, in o c u la te d with Glomus f a s c i c u l a t u s (myco), amended wit h both Glomus f a s c i c u l a t u s and phosphorus, and unamended c o n t r o l , w ith and w ith out the nematode Meloidogyne h a p l a . o o U) O CD —i 00-1 0 CONTROL a CONTROL/NEMA CO-i P/NEMA X MYCO X MYCO/NEMA "* M/P/NEMA I o x 200 CM — Ld QL m 3 ° - CO □Q LjJ o 160 ~1 o ' 280 160 JULIAN DAY Figure 33- 280 160 280 fc i V J1 M Figure 31*. Fresh shoot weight of Allium cepa supplemented with phosphorus, in o c u la te d with Glomus f a s c i c u l a t u s (myco), amended with both Glomus f a s c i c u l a t u s and phosphorus, and unamended c o n t r o l , wit h and w ith out th e nematode Meloidogyne h a p l a . O A CONTROL CONTROL/NEMA 0 o - * X 2“ X .. * P/NEMA MYCO MYCO/NEMA M/P/NEM A } vn w o to o IO U1 160 250 160 JULIAN DAY Figure 31*- 250 o 160 250 154 fertilized with P a t transplant (Figure 35). Plants fertilized with P tended to support more nem atodes than control plants and few er nem atodes than plants receiving VAM. Oviposition by M. hapla fem ales was first observed on 8/5/82, ca two months a fte r transplanting. There was no significant difference in nem atode levels betw een sampling dates for any tre a tm e n t, indicating th a t only one nem atode generation invaded root tissue during the experim ent. Levels of M. hapla juveniles in the soil decreased on the first th ree sample dates, 6/28-8/5/82, and then increased significantly (P=0.05) from th e third until the final sample date, 8/5-9/16/82 (Figure 36). Nematode density was g reatest in m icroplots inoculated with VAM alone and lowest in m icroplots containing control plants. The index of change in M. hapla density (Pj/P.) for the VAM, VAM + P, P, and control treatm en ts was 18.8, 3.0, 0.7, and 0.1, respectively. Levels of G. fasciculatus spores in the soil significantly (P=0.05) decreased, increased, and decreased again over the course of th e growing season (Figure 37). There were no significant differences in spore levels for those treatm en ts inoculated with G. fasciculatus. Some indigenous spores w ere present in m icroplots not inoculated with G. fasciculatus. The density of spores in non­ inoculated m icroplots was less than th a t of inoculated microplots, but followed the same general trends over the course of the growing season. DISCUSSION D ata from the field and greenhouse experim ents suggest th a t mycorrhizal colonization may benefit A. cepa infected by M. hapla. Fresh bulb weight was similar for mycorrhizal plants grown in m icroplots and fertilized a t transplant with P and non-m ycorrhizal plants supplemented with P (Figure 33) even 155 Control Phosphorus LO LO- O- in MC 160 220 280 JULIAN DAYS Figure 35. Number of Meloldogyne hapla in 0.5 gram of roots of A11iurn cepa supplemented with phosphorus, in o c u la te d with • Glomus f a s c i c u l a t u s , o r amended with both Glomus f a s c i c u l a t u s and phosphorus, and unamended c o n t r o l . 156 0 Meloidoffyne hapla X Ifeloidogyne hapla + Glomus fasiculatus ioH oH o WEEKS Figure 36. Number of Meloldogyne hapla j u v e n i l e s In 100 cm^ o f s o i l surrounding A i 1iurn cepa supplemented with phosphorus, inoc ulated with Glomus f a s c i c u l a t u s . amended with both Glomus f a s c i c u l a t u s and phosphorus, and unamended c o n t r o l . 157 O in - o (/) N* © C o n tro l a C o n tro l/N o m a □ P h o a p h o ru o P h o o p h o ru a /N o m o X E o X M y c o n titz a * M y c o n W z a o /N o m a M y c o /P h o o /N o m a o o to a: Ul O a. to CO 3 3 a •*» a (0 O ' ti w 6 o z < Ul 160 220 280 JULIAN DAY Figure 37* 3 Number of Glomus f a s c i c u l a t u s spores in 100 cm s o i l surrounding A1 Hum cepa supplemented with phosphorus, inoculat ed with Glomus f a s c i c u l a t u s (mycorrhizae), amended with both Glomus f a s c i c u l a t u s and phosphorus, and unamended c o n t r o l , with and w ith out th e nematode Heloidogyne hapla. 158 though nem atode densities were twofold g reater in the mycorrhizal plants (Figure 35). These results indicate th a t A. cepa w ith an established VAM association was more to leran t to M. hapla parasitism . Conversely, VAM did not benefit A. cepa infected by M. hapla under greenhouse conditions. In this experim ent, nem atodes were established in plants before a VAM association was initiated. Nematode density averaged c a 83 M. hapla/root system for the VAMinoculated plants and was perhaps sufficient to prevent a recovery in growth due to colonization by th e fungus. The presence of M. hapla within A. cepa roots did not seem to influence the production of VAM spores, as indicated by th e sim ilarity in spore densities in soil surrounding nem atode-infected and non-infected plants both in the green­ house and microplot experim ents (Figures 32, 37). D ata from th e greenhouse experim ent suggests, however, th a t the infectivity of spores may have been decreased by the presence of nem atodes, since more spores remained in the soil surrounding nem atode-infected plants a t the end of the experim ent (Figure 32). Results from the greenhouse experim ent indicate th a t VAM colonization influences the ontogeny of M. hapla. The number of nem atodes within roots was very similar in plants inoculated with the nem atode alone or with the nem atode and fungus for the first six weeks of the experim ent. This is not surprising, since a VAM association was not established when these nem atodes entered root tissue. Eight weeks a fte r planting, th ere was an increase in nem atode density on both VAM -treated and non-treated plants which, due to th e appearance of eggs in week six, would appear to be due to an influx o f second generation nem atodes. The decline in second-stage juveniles within th e roots of mycorrhizal plants from week six to week eight and the low percentage of adult fem ales observed, 159 however, suggest th a t the nem atode increase associated with this tre atm en t was due to sampling error rath er than to a true change in nem atode population levels. Conversely, the data indicate th a t nem atode levels were increased in nonm ycorrhizal plants due to reproduction. More than half the nem atodes observed in non-mycorrhizal plants six weeks a fte r planting had m atured to the adult fem ale stage. Although the difference in th e numbers of second-stage juveniles observed in those plants was not different betw een weeks six and eight, it is highly likely th a t some influx of nem atodes occurred, since th e average nem atode in the second-juvenile stage a t week six would have progressed to the next developm ental stage in ca two weeks (Tyler, 1933). In the microplot experim ent, significantly (P=0.Q5) few er nem atodes pene­ tra te d mycorrhizal plants fertilized a t transplant w ith P than m ycorrhizal plants not fertilized (Figure 35). Graham e t al. (1981) reported th a t P nutrition associated with VAM decreased root perm eability and the exudation of root m etabolites into the rhizosphere. The a ttra c tio n of nem atodes to root exudates may thus be m ediated by P levels in root tissue. It is possible th a t th e P benefits normally gained by a VAM association were negated by damage to th e roots and fungal network in the transplanting process. Supplementing mycorrhizal plants with superphosphate may have restored root P levels so th a t the host-finding behavior or the penetration of roots by M. hapla was a lte red . From these d ata, the failure of nem atodes to en ter or become established in control plants cannot be a ttrib u ted to any particular facto r. It is possible th a t control plants were stressed to the point th a t nem atodes entered roots and exited im m ediately. The tim e-fram e of the experim ent, however, was not adequate to confirm this possibility. 160 In the greenhouse experim ent and in prelim inary studies, VAM colonization seemed to a ffe c t the population growth but not the pathogenicity of M. hapla. In the microplot experim ent, VAM colonization alone did not lim it M. hapla density, but conferred a tolerance to plants for M. hapla parasitism . The differential ability of mycorrhizal plants to support and a lte r th e pathogenicity of M. hapla in th e greenhouse and microplot environm ents may have been due to differences in the sequence of root colonization by nem atodes and fungi or to the disruption of the root system during transplanting. Soil populations of M. hapla increased only threefold over the course of the growing season in m icroplots inoculated with VAM + P, as compared to a 19x increase for microplots inoculated with VAM alone. The magnitude of the difference in M. hapla density betw een these treatm en ts suggests th a t some unidentified factor or factors influenced the population dynamics of M. hapla in this study and illustrate the complexity of th e association. Conflicting reports in the lite ra tu re concerning the influence of VAM on nem atodes may not be due to the specificity of plant-nematode-VAM system s as commonly reported but rath er to differences in experim ental methodology or environm ental conditions. Addi­ tional inform ation on the variables m ediating th e response of plants and nem atodes to VAM is required before a tru e understanding of the interactions betw een these organisms can be realized. Influence of Glomus fasciculatus on the Location and Penetration of Allium cepa Roots by Meloidogyne hapla INTRODUCTION The growth of some phytoparasitic nem atode populations is limited by the presence of vesicular-arbuscular m ycorrhizae (VAM) in th e host plant (Kellam and Schenck, 1980; Sitaram aiah and Sikora, 1980). Both juvenile and adult nem atodes are reportedly influenced by VAM. Sikora (1979) proposed th a t VAM colonization a ffe cts the penetration of roots by juveniles and the development and m aturation of nem atodes within roots. The objective of this study was to examine th e influence of VAM colonization on the ability of second-stage Meloidogyne hapla juveniles to locate and penetrate roots o f A. cepa. Several studies support Sikora's proposal th a t the ability of nem atodes to p en etrate root tisses is reduced in the presence of m ycorrhizae (Cooper, 1981; Sitaram aiah and Sikora, 1980). The hypothesis th a t the host-finding behavior of nem atodes is adversely a ffe cted by mycorrhizal colonization has been supported but not examined in detail. Two experim ents are reported here. One experim ent was conducted to assess the ability of nem atodes to p en etrate mycorrhizal roots when only limited host-searching behavior is required. The relativ e attractiv en ess of mycorrhizal and non-mycorrhizal root systems tc nem atodes in search of a host plant was examined in a second experim ent. MATERIALS AND METHODS Experim ent 1. Penetration of A. cepa by M. hapla placed in close proximity to the root system. 162 Krummery Special onions were sown into pots (d = 6.40 cm) containing 342 g of a pasteurized mineral soil with a pH of 3.9 and a P level of 1.1 ppm. At the tim e of planting, one of three soil treatm en ts was adm inistered in 3 ml of w ater to each pot: w ater alone, 2000 spores of the VAM Glomus fasciculatus. or 9.13 mg KHjPO^ to add 7 ppm P to the soil. The level of P fertilizer used had been predeterm ined to stim ulate onion growth com parable to VAM colonization. Pots fertilized and unfertilized were supplemented with NH^NO^ and KNO^, respect­ ively, so th a t the only nutrient th a t differed betw een tre a tm e n ts was P. All pots, thinned to one seedling one week a fte r planting, were immersed in a w ater bath maintained a t 21 C and w atered daily. One month a fte r seeding, 300 M. hapla second-stage juveniles in 3 ml of w ater were inoculated into th ree small holes around the base of each plant. Two, four, eight, and 12 days la te r, six pots from each treatm en t were destructively sampled. The plants were gently removed from the soil, washed, and weighed. The en tire root system was stained in lactophenol with 0.01% acid fuchsin and the nem atodes within were enum erat­ ed under a dissecting microscope. This experim ent was repeated once using a muck soil and the same methodology. Experim ent 2. Location and penetration of A. cepa roots by M. hapla. Krummery Special onions were sown into pots containing 63.10 g of a pasteurized 7:1 sand/muck soil with a pH of 6.6 and P level of 4.8 ppm. One of three treatm ents were adm inistered in 5 ml of w ater to each pot as described previously: w ater alone, 2000 spores of G. fasciculatus, or 12.9 mg K ^ P O ^ to add 43 ppm P to the soil. The pots were maintained in a greenhouse and w atered daily. A fter one month, the seedlings were transplanted to 23.3 cm lengths of 163 vinyl pipe (d=7.5 cm) split longitudinally and sealed a t each end with tap e. There were three holes in the bottom of each tube for w ater drainage. All six possible combinations of plants from th e three soil treatm en ts were represented: C-M, C -P, P-M, C -C , P-P, M-M. One week a fte r transplanting, 800 M. hapla secondstage juveniles in 5 ml of w ater were added to a single depression in the middle of each tube, equidistant to the two plants. Two, four, eight, and 12 days later, six tubes representing each of the six treatm en ts were harvested. The cum ulative degree hours (base = 9 C) for each harvest were 534, 1203, 2705, and 4162 degree hours (DH). Five cm of soil in the middle of each tube was discarded. The remaining 10.25 cm of soil a t each end of th e tubes and the onion plants were then carefully removed. The plants were washed, weighed, and the root systems cut into small pieces and placed into flasks containing 50 ml of a solution of ethoxyethyl m ercuric chloride and dihydrostreptomycin sulfate. The flasks were ag itated on a gyratory shaker for two days, a fte r which tim e the solution was poured through a 400 pm mesh screen and collected for nematode observation. One hundred cm of soil surrounding each plant was assayed by a modified sugar flotation-cetrifugation technique (Jenkins, 1964). The d ata were analyzed as if each combination of original treatm en ts constituted one tre a tm e n t. For example, control plants grown in the same tube with mycorrhizal plants were designated Cm and tre a ted separately from control plants grown with P -fertilized plants, C^. This classification scheme was designed to dem onstrate the relative differences in the attractiv en ess of control, P-fertilized, and VAM-inoculated plants. An analysis of variance was perform ed on the data using all nine tre a tm e n t combinations: Cm, Mc , Pc , Cp, Mp» Pm’ Cc ’ Pp, and The Wilcoxin signed rank te s t was used to evaluate sets of paired 164 observations (P -C , M -P . C -M ) on the number of M. hapla in soil and root c p p m m c — —t— systems. RESULTS Experim ent 1. Significantly (P=0.05) few er M. hapla p enetrated mycor­ rhizal plants than plants amended with phosphorus or unamended plants 12 days following inoculation (Figure 38). Nematode density increased (P=0.01) within each treatm en t during the course of the experim ent (Table 30). Similar trends were observed when nematode levels were considered on a per gram, centim eter, or root tip basis (Figure 39). There were few er M. hapla per 0.1 gram root and root tip in mycorrhizal plants two days following inoculation. There were no differences in root length or weight betw een treatm en ts for any sample date, although values for both growth param eters increased within each treatm en t in the 12 days following th e inoculation of plants with M. hapla. An adjustm ent of nem atode density according to root length by an analysis of covariance produced the same results as the analysis of variance in Table 30. Experiment 2. There were no significant (P=0.05) differences in the number of nematodes observed in th e roots and soil surrounding A. cepa tre a ted with P, VAM, or control plants when all nine tre a tm e n t combinations were considered in an analysis of variance. When th e d ata w ere paired according to the arrangem ent of plants within the tubes, however, significantly (P=0.05) more nem atodes were found in th e soil around P -fertilized plants four, eight, and 12 days following inoculation when forced to choose betw een the P -fertilized and VAM-treated plants (P vs M ) (Table 31). The difference in nem atode density r in and surrounding A. cepa were not statistically significant (P=0.05) for any 165 © Water Control x o I— Phosphorus Control m Mycorrhizae a. LSD.05 = 42.74 LJ CM O, DAYS AFTER INOCULATION Figure 38. Number o f Meloidogyne hapla w i t h i n r o ot systems of A11iurn cepa supplemented with phosphorus (phosphorus c o n t r o l ) , in o c ula ted with Glomus f a s c i c u l a t u s (mycorrhizae), or unamended (water c o n t r o l ) . 166 Table 30. Analysis o f va ri a n c e f o r Meloidogyne hapla d e n s i t y w it h in A11ium cepa t r e a t e d with Glomus f a s c i c u l a t u s . phosphorus, o r tap w at er. Source DF Sum o f squares Mean square F Sample d a te 3 b Treatment 3 73455.0 24485.0 17.87 ** 2 11360.0 5680.0 4.15 * Date x trea tme nt 6 5802.0 967.0 Error 60 82216.0 1370.0 Total 71 172830.0 0.71 P l a n t s were d e s t r u c t i v e l y sampled 2, *4, 7, and 12 days following i n o c u l a t i o n . P l a n t s were t r e a t e d with mycorrhizae, phosphorus, or tap wa ter. Iog ,0 (M. hapla/A g ROOT) log10 ( M. hapla / c m log10 (AT. hapla/ROOT TIP + 1) ROOT + 1) L S D . = 0 .1 5 4 9 to to 0 M ycorrhizae □ M ycorrhizae x P h o sp h o ru s C ontrol x P h o sp h o ru s C ontrol 0 C ontrol 0 C ontrol to o L S D . = 0 .4 8 3 6 0 M ycorrhizae x P h o sp h o ru s Control O Control o L S D . = 0 .2 7 1 0 o o . DAYS AFTER INOCULATION Figure 39. DAYS AFTER INOCULATION T T DAYS AFTER INOCULATION Number of Meloidogyne hapla per c e n t i m e t e r , 0.1 gram, o r r oot t i p in the root systems of A11 iurn cepa supplemented with phosphorus (phosphorus c o n t r o l ) , i n oc ula te d with Glomus f a s c i c u l a t u s (mycorrhizae), and unamended c o n t r o l . 168 Table 31. Summary of the d i f f e r e n c e s in the number of Meloldogyne hapla observed In s o i l samples from A111 urn cepa inocu la ted with Glomus f a s c i c u l a t u s , amended with phosphorus, or noninoculated and unamended. Days a f t e r i n o c u l a t i o n 2 days k days 8 days 12 days C > M3 m c Cm< Mcr C >M m c Cm< m Mrc C -te P c C < P P c C > P c * Pr P >M m p P >M m p P P * P c P >M m p * P >M m p a C apital l e t t e r r e f e r s to trea tme nt (C = c o n t r o l : f a s c i c u l a t u s : P ■ + phosphorus) S ubs c rip t r e f e r s to tr ea tm en t o f the o t h e r same c o n t a i n e r . c * M ■ + Glomus p l a n t grown in the D iff ere nc es between the pa ire d da ta a r e s i g n i f i c a n t according to Wilc oxi n's Signed Rank T e s t. (P =0.05) 169 other combination of treatm en ts (M„ _ or P„c vs C p ). In tubes containing° a c vs C m control plant and a VAM-inoculated plant, th ere was a tendency for higher soil levels of M. hapla to be associated with the control plant two and eight days a fte r inoculation and with the mycorrhizal plant four and 12 days a fte r inoculation. Levels of M. hapla in th e roots of A. cepa were low for all treatm en ts. Many nem atodes observed in root fragm ents w ere evidently not expelled by the rotary shaker method used in this experim ent. Control plants visually appeared to have smaller root and shoot systems than VAM-treated plants, which in turn tended to be smaller than P-fertilized plants (Table 32). The slight discrepancy in root weight betw een the tre atm en ts was consistent but not statistically significant (P=0.05). Shoot weight was significantly (P=0.05) g re a ter in P -fertilized plants grown with VAM-inoculated plants as compared to the other tre a tm e n t combinations four days a fte r nem atode inoculation. Eight days following inoculation, VAM-treated plants grown with control plants had shoot system s significantly (P=0.05) larger than any other treatm en t. DISCUSSION Fewer M. hapla entered roots of A. cepa when a VAM association was present (Figures 38, 39). Preliminary experim ents similar to the one described here produced similar results. The detrim ental influence of mycorrhizal colonization on th e penetration of roots by nem atodes was also reported previously by other authors. Cooper (1981) found th a t M. incognita did not enter roots with g re a ter than 10% levels of m ycorrhizal colonization. Similarity, Table 32. Root and shoot f r e s h weights (g) o f A l l i um cepa i no c ul a te d with Glomus f a s c i c u l a t u s . amended with phosphorus, and noninoculated and unamended c o n t r o l . Day 2 Treatment3 Day 4 Day 12 Root Shoot Root Cm 0.0950 ab 0.1317 a 0.0633 a 0.1417 ab 0.1017 a 0.1750 ab Mc 0.0750 ab 0.1550 a 0.0667 a 0.1533 ab 0.1083 a 0.3783 c 0.0800 ab 0.1367 a 0.0583 a 0.1050 a 0.1050 a 0.1400 a P c 0.0730 ab 0.1183 a 0.0800 a 0.1250 ab 0.1200 a 0.1450 ab P m 0.0760 ab 0.1300 a 0.0833 a 0.1117 a 0.1217 a 0.2050 ab M P 0.0800 ab 0.1150 a 0.0867 ab 0.2167 c 0.1050 a 0.2567 b C c 0.0783 ab 0.1150 a 0.0883 ab 0.1233 ab 0.1433 b 0.2117 ab P P 0.1150 b 0.1667 a 0.1117 b 0.1850 be 0.0783 a 0.1600 ab Mm 0.0600 a 0.1200 a 0.0850 ab 0.1375 ab 0.0650 a 0.2000 ab CP Shoot Root Shoot Values w i t h i n a column followed by the same l e t t e r a r e s i g n i f i c a n t l y (P = 0.05) d i f f e r e n t according to Duncan's M u lt ip le Range T e s t . a C ap it al l e t t e r = tr e a tm e n t (C = c o n t r o l : M = + Glomus f a s c i c u l a t u s : S u b s c r i p t = tr ea tm en t o f the o t h e r p l a n t grown in the same c o n t a i n e r P = + phosphorus) 171 Sitaram aiah and Sikora (1980) reported th a t the penetration of Rotylenchulus reniform is into cotton was impeded in th e presence of VAM. responsible for this phenomenon has not been identified. The mechanism Sim iiarities in the numbers of nem atodes penetrating P-fertilized and VAM-inocuiated A. cepa two and four days a fte r inoculation suggest th a t some tr a it shared by these plants, perhaps root P levels, was involved. Results from this study may provide some insight for findings th a t VAM fungi inoculated slightly prior to or simultaneously with nem atodes have no noticeable influence on nematode population level for several weeks. Kellam and Schenck (1980) found no significant difference in the number of galls produced by M. incognita on soybean until 12 weeks following inoculation of the nem atode and fungus. Similarly, Sikora and Schonbeck (1975) reported th a t differences in M. hapla density in mycorrhizal and non-m ycorrhizal c a rro t roots were g re a ter 12 weeks following inoculation than a t six weeks. In the studies referred to above, m ycorrhizal colonization may not have been sufficiently established to inhibit root penetration by inoculated nem atodes. The progeny of inoculated nem atodes, however, faced with th e task of invading root system s with a well-developed VAM association, may have been impeded in th e ir effo rts to enter roots. Although the reduced nem atode levels in these studies may also be due to the decreased survival or delayed developm ent of nem atodes in mycorrhizal roots, it seems unlikely th a t VAM colonization would not have been sufficiently established to also a ffe c t population levels of th e initial nem atode generation. Evidence th a t the ability of nem atodes to locate and p en etrate roots is reduced in th e presence of VAM should be considered when interpreting d ata 172 from nematode/VAM interaction studies. For exam ple, Schenck e t al. (1975) concluded from levels of M. incognita juveniles in the soil th a t nem atode populations were increased in th e presence o f m ycorrhizae. Results from my study dem onstrate th a t high levels of juveniles in th e soil may also be due to th e reduced ability of nem atodes to en ter plant roots. Schenck e t al.'s conclusions may have been d ifferen t had they assessed the to ta l nem atode density in soil and roots. C ertainly not all reports of increased nem atode levels in mycorrhizal plants are due to the erroneous m easurem ent of the nem atode population. The to ta l root biomass available for nematode penetration, nem atode inoculum levels, and the density of nem atodes established within roots may modify the influence o f VAM on the penetration process. The hypothesis th a t M. hapla is differentially a ttra c te d to mycorrhizal and non-mycorrhizal A. cepa was supported but not confirm ed (Table 31), since the experim ent presented here provided only a gross indication of the direction nem atodes moved to locate a host. U nfortunately, the failure of the shaker technique to e x tra c t M. hapla from the roots of A. cepa made in terpretation of the data difficult. Decreased nem atode levels in the soil could not be confidently a ttrib u ted to th e failure of nem atodes to m igrate towards a plant since the number of nem atodes successfully penetrating roots was not known. M. hapla seemingly preferred P-fertilized A. cepa over mycorrhizal plants, as evidenced by the paired difference te st. It is possible th a t mycorrhizal plants possessed (or lacked) some a ttrib u te making them less a ttra c tiv e to M. hapla. Size of the root system did not seem to be an im portant facto r since root weights of mycorrhizal plants were not different from th e other plants. R ecent studies dem onstrated th a t root membrane perm eability is influ­ enced by the P levels within root tissue (Ratnayake e t al., 197S; Graham e t al., 173 1981). It is possible th a t greater amounts of root exudates are released by roots low in P, which are im portant stimuli a ttra ctin g nem atodes to a host plant (Prot, 1980). It may be th a t root P levels differed dram atically between treatm ents in this experim ent and contributed to the d ifferential attractiv en ess of roots to M. hapla. Since root P levels were not measured, th e magnitude of th e differences betw een the treatm ents a re not known, although it seems likely th a t all Pfertilized plants had higher root P levels than control plants. The inability of nem atodes to consistently choose control plants, therefore, suggests th a t other plant a ttrib u te s are also involved in the host-finding behavior of M. hapla or th a t different types of stimuli are involved in th e long- and short-range location of roots by nem atodes. For example, it is possible th a t CC^ gradients proportional to plant size are the initial stimuli a ttra ctin g nem atodes and th a t root exudates or other stimuli influence nematode movement to a specific location on the root surface. Until more is known concerning the host locating behavior of nematodes, the influence of VAM on the attra ctio n of nematodes to plants cannot be fully determ ined. Influence of Glomus fasciculatus on the Stage-Specific Development and Reproduction of Meloidogyne hapla Inhabiting Roots o f Allium cepa INTRODUCTION The ontogeny of Meloidogyne spp. within plant roots has been described in detail (Bird, 1959; Triantaphyilou and Hirschmann, 1960). Feeding, sexual differentiation, and growth of these nem atodes commence in the second juvenile stage. The third- and fourth-juvenile stages are devoted to sexual development in the absence of feeding or increased size, and occur in relatively rapid succession. A fter th e final m olt to th e adult stage and prior to oviposition, fem ale nematodes enter a period of feeding and growth. R ates of developm ent for the juvenile and pre-ovipositional adult stages are closely related to tem perature (Davide and Triantaphyilou, 1967; Wong and Mai, 1973a; Tyler, 1933; Vrain e t al., 1978) and are modified by attrib u te s of the host plant (Ferris and Hunt, 1979; Griffin and Hunt, 1972). The im portance of food in the developm ental process suggests th a t com petition with concom itant organisms for plant resources may also alter the developm ental ra te of some life stages of Meloidogyne spp. My studies on th e interaction betw een vesicular-arbuscular m ycorrhizae (VAM) and M. hapla within roots of Allium cepa support this hypothesis. In one experim ent, for exam ple, over tw ice (P=0.08) as many adult M. hapla were observed in A. cepa inoculated with the VAM, Glomus fasciculatus. even though the to ta l number of nem atodes per root system was not different between non-mycorrhizal and m ycorrhizal plants. The objective of this study was to examine the influence of mycorrhizal colonization on the developmental rates of the juvenile and adult stages of M. 17** 175 hapla infecting A. cepa. Due to the genetic variability in individuals, both the fa stest and median developm ental rates of nem atodes were considered. The slowest developmental tim e was not calculated for any nem atode stage, since dead nem atodes could not be accurately distinguished from living nem atodes within root tissue. MATERIALS AND METHODS Onions (cv Krummery Special) were sown in com partm ents of plastic nursery trays containing 217 g (dry wt) of a 7:1 mix of sand and a Houghton muck soil with a pH of 6.5 and a soil P level of 1.3 ppm. One third of the trays were inoculated ca 3 cm below the soil surface with 1600 spores of the mycorrhizal fungus Glomus fasciculatus in 5 ml of w ater. Another third of the trays were fertilized with 0§H 00 o o Ld 6 o j —j r ID < Ui 8000 11000 ~1 14000 DEGREE HOURS (b a s e 9 C) Figure k j . Number o f eggs produced per o v i p o s i t i n g Meioidogyne hapla female i n h a b i t i n g Ai Hum cepa supplemented with phosphorus, in oc ul a te d with Glomus f a s c l c u l a t u s (m yco rrhizae), and unamended c o n t r o l . 189 <£> O □ Mycorrhizae X Phosphorus Control O Control 9 t U3 9 * O O) f— • - I THIRD » FOURTH STAGES COMBINED FEM - PREOVIPOSITING FEMALES FEMDENS - NEMA OENSITY FACTOR INFLUENCING EGG PRODUCTION NEMSITE * NO. FEEDING S IT E S AVAILABLE FOR NEMA PENETRATION OVIFEM - OVIPOSITING FEMALES OVITEMP - TEMP FACTOR INFLUENCING EGG PRODUCTION OWEGG - OVER-WINTERING EGGS (USER INPUT) PENLARV - LARVAE WHICH HAVE PENETRATED ROOT (PRECEDES E L2) SOILARV - SECOND-STAGE JUVENILES IN THE SOIL SURV(STAGE) - SURVIVAL RATE FOR PARTICULAR STAGE TEMPEN - TEMP FACTOR INFLUENCING ROOT PENETRATION VIABLE - ARRAY NAME USED IN TEMPEN VIALARV - VIABLE LARVAE - NO. AVAILABLE TO PENETRATE SUBROUTINE NEMA 35 COMMON/DRIVE/DT. LX. NUMDAY.TEMP. OWEGG. BASE. OH COMMON/ONION/NEMSITE.TQTSITE.NEWROOT. ROOTWT COMMON/NEMA/EGG. SOILARV. E L 2 . L L 2 . L 3 L 4 . FEM.OVIFEM. TOTNEM COMMON/NEMFEED/NEMAFED. FEEDNEM 40 C c REAL L 1 .E L 2 .L L 2 .L 3 L 4 .N E M S IT E N O OO DIMENSION X1(10).X2(10).X3{10).X4<10),X5(10).X6(10),X7(10) DIMENSION VIABLE(11) DIMENSION STORE(IO) DIMENSION AUX(tO) DATA VIABLE/.Ol,.05..10..25..50..75.1.00..75..50..25..01/ 45 SO 55 C C INITIALIZE VARIABLES C IF(LX.OT.O) GO TO 100 C DO 5 1-1.10 x i(i)« o .o 60 5 C 65 70 75 80 100 X2(1)-0.0 X3(I)-0.0 X4(I)*0.0 X5(I)-0.0 X6(I)-0.0 X7(I)-0.0 CONTINUE EGG-0.0 SOILARV-O.O TOTGONE-0.0 EL2-0.0 LL2-0.0 L3L4-0.0 FEM-O.O OVIFEM-O.O FEMDENS-1.0 0VITEMP-1.O SURVSL - 0.0 SLL2 - 0.0 SL3L4 - 0.0 SFEM - 0.0 SOFEM - 0.0 SURVX1- 1.00 PENLARV - 0.0 AXTOT1-0.0 AXT0T2-0. CONTINUE 85 C CALC. EGG FROM OVERWINTERING EGGS (1ST ITERATION ONLY) AND C+ OVIPOSITING FEMALES (14 EGGS/FEMALE/OAY AT OPTIMAL TEMP AND DENSITY) C C ^ O 90 EGG- (DT* (OWEGG ♦ 0VIFEM*14.)) - FEMOENS * OVITEMP OWEGG-O.O 95 100 105 110 120 125 130 135 210 115 C C CALC. OEV.RATE FOR EGG STAGE BASED ON TEMP C C IF THEN IF(TEMP.LT.10) GO TO 200 DEVEGG-(-0.0564 + 0.00564-TEMP) - DT GO TO 300 C ELSE 200 OEVEGG-O.OOOI 300 CONTINUE C END IF C CALL MODEL1(XI.STORE.DEVEGG.AXTOT1.L 1) PRINT-.-EGG - *,LI C C ADD EGG INPUT TO EGG DEVELOPMENT PROGRAM C X1(1)-X1(1) - EGG • 0.30 C C KILL OFF 70 PERCENT OF ALL EGGS - GUESSTIMATE C C TEMP FACTOR FOR SOILARV MOBILITY C TEMPEN - FNL(VIABLE,10..2.,10.TEMP) C C CALC. NO. OF LARVAE CAPABLE OF PENETRATING ROOTS BASED ON TEMP C SOIL-SOILARV-TOTGONE IF(SOIL.LT.O.) SOIL"0.0 IF(TEMP-20.)400,400.500 400 DIES0IL-(S0IL*0.142857/2)*DT GO TO 700 500 DIES0IL-(S0IL-0.142857)*DT GO TO 700 700 CONTINUE AVAIL-(SOIL-OIESOIL)-TEMPEN IF(AVAIL-NEMSITE>800.900.900 800 PENLARV-AVAIL GO TO 1000 900 PENLARV-NEMSITE 1000 CONTINUE GONE-D1ESOIL+PENLARV TOTGONE-TOTGONE + GONE 140 145 150 155 160 165 170 C C C CALC. RATE OF SOILARV OEV C THE DEVELOPMENT PROGRAM IS USED HERE TO PROGRESS NEMAS C+TO DEATH. ONLV NEMAS STILL IN DEVELOPMENT ARE USED TO CALC. THE C+THE NO. OF LARVAE CAPABLE OF PENETRATING ROOTS C SOILARV DIE IN 14 DAYS IF TEMP LT 20 AND 7 DAYS IF TEMP GT 20 C SOILARV DIE AT VERY SLOW RATE (NEGLIGIBLE) WHEN TEMP IS LT 9 C IF(TEMP-20.)1020.1020,1030 1020 DECSOIL • (0.142857/2.) * OT GO TO 700 1030 DECSOIL - 0.142857 * DT GO TO 1050 1050 CONTINUE C C SURVIVAL RATE OF SOILARV C C CALL MODEL3(SOILARV.STORE.OEVEGG.DECSOIL.SURVSL) PRINT*.'SOILARV * '.SOILARV C C CALC. OEV RATE FOR PENETRATED LARVAE - EARLY 2ND-STAGE C IF(TEMP.LT.BASE) GO TO 1300 IF(TEMP-25.) 1100,1200.1200 1100 0EVEL2 * OT * (0.10/(18192.04377 ♦ TEMP ** (-3.04976))) GO TO 1400 1200 DEVEL2- 0.10 • OT GO TO 1400 1300 DEVEL2-0.0001 1400 CONTINUE V* CALL MODEL1(X3.AUX.DEVEL2.AXT0T2.EL2) PRINT*,'EL2 * *,EL2 C X3(1)»PENLARV C C CALC DEV RATE FOR LATE 2NO STAGE JUVENILES C IF(TEMP.LT.BASE) GO TO 1700 IF(TEMP-25.) 1500.1600.1600 1500 0EVLL2 ■ DT • (O.167/(TEMP/(-16.538 ♦ 1.72811 * TEMP))) IF((TEMP.GE.9.).AND.(TEMP.LT.11.)) DEVLL2-0.05 • OT GO TO 1800 1600 DEVLL2-0.167 * OT GO TO 1800 1700 DEVLL2-0.0001 1800 CONTINUE C SURVLL2-1.00 C CALL MOOEL2(DEVEL2.DEVLL2.AUX.X4.SURVLL2,SLL2.LL2) PRINT*,"LL2 - *.LL2 C C CALC. OEV RATE FOR 3RD AND 4TH STAGE JUVENILES (COMBINED) C IF(TEMP.LT.BASE) GO TO 2100 IF(TEMP-25.) 1900.2000.2000 1900 DEVL3L4 ■ OT * (0.50/(8.756129 • EXP(-0.08549 • TEMP))) GO TO 2200 2000 DEVL3L4-O.SO • DT GO TO 2200 2100 DEVL3L4-0.0001 2200 CONTINUE SURVL34-1.00 C CALL MODEL2(DEVLL2.0EVL3L4.AUX.X5.SURVL34.SL3L4.L3L4) C C CALC. OEV RATE OF PRE-OVIPOSITING FEMALES C 210 2300 2400 215 2500 2600 IF(TEMP.LT.BASE) €0 TO 2500 IF(TEMP-25.) 2300.2400.2400 DEVFEM - DT * (0.125/(4.01124 - 0.1177928 * TEMP)) GO TO 2600 DEVFEM*0.125 * DT GO TO 2600 DEVFEM-O.OOOt CONTINUE SURV0VI-1.00 C 220 225 230 235 240 245 CALL M0DEL2(0EVL3L4.DEVFEM,AUX,X 6 .SURVOVI,SFEM.FEM) C C CALC. OEV RATE OF OVIPOSITING FEMALES • DEVELOPMENT LEADS TO DEATH C IF(TEMP-25.) 2700.2800.2800 2700 DEVOFEM - (0.02/2.) * OT GO TO 3000 2800 DEVOFEM ■ 0.02 • DT 3000 CONTINUE C DEADFEM-1.00 0ECFEM-.O5 C CALL M0DEL3(0VIFEM.AUX.DEVFEM,DECFEM.DEAOFEM) C C CALC. EFFECT OF NEMA DENSITY ON EGG PRODUCTION C FEM0ENS-1.00 C C CALC. EFFECT OF TEMP ON EGG PRODUCTION C 0VITEMP-1.00 C C CALC. TOTAL NO. OF NEMAS OCCUPYING ROOT C TOTNEM - EL2 ♦ LL2 ♦ L3L4 + FEM + OVIFEM C RETURN END SUBROUTINE NEMFEED 1 C C C »* 5 C C *•**• 10 IS 20 C C C c c c c c c c C C C C r* w c VARIABLES: DH « DEGREE HRS FOR THIS ITERATION • FEEOFAC- INFLUENCE OF NEMATODE DENSITY ON THE FEEDING FUNCTION •+ OF ONE NEMATODE (EXPRESSED AS PERCENT GROWTH REDUCTION) * FEEDNEM- TOTAL NO. OF NEMATODES INHABITING THE ROOT SYSTEM EXCEPT L3L4 STAGE JUVENILES * FEM- STATE VARIABLE: PRE-0VIP0SIT1NG FEMALE STAGE • LL2- STATE VARIABLE: LATE 2ND STAGE ♦ L3L4- STATE VARIABLE: 3RD.4TH STAGE (COMBINED) * NEMAFED- PERCENT GROWTH REDUCTION RESULTING FROM NEMA FEEDING * OVIFEM- STATE VARIABLE: OVIPOSITING FEMALE STAGE PERNEMA - FEEDING EFFECT OF EACH NEMA DEPENDENT ON NEMA DENSITY * SOILARV- STATE VARIABLE: 2ND STAGE JUVENILES IN SOIL X3- STATE VARIABLE: EARLY 2ND STAGE JUVENILES IN ROOT * * * * * 25 C SUBROUTINE NEMFEED C COMMON/DRIVE/DT.LX.NUMDAY.TEMP.OWEGG.BASE.OH COMMON/ONION/NEMSITE,TOTSITE.NEWROOT.ROOTWT COMMON/NEMA/EGG.SOILARV.EL2.LL2.L3L4.FEM.OVIFEM.TOTNEM COMMON/NEMFEED/NEMAFED.FEEDNEM 30 C 35 REAL NEMAFE0.LL2 C C NO. OF NEMATOOES FEEDING WITHIN ROOT SYSTEM C FEEDNEM - EL2 + LL2 + FEM + OVIFEM IF(FEEDNEM.LT.1.) GO TO 100 c C CALC. FEEDING FACTOR BASED ON NEMATODE DENSITY C IF(DH-8000.) 40,40.50 40 PERNEMA - (0.0005019 * 0.65623/FEEDNEM) • OT GO TO 60 50 PERNEMA • ((0.0005019 * 0.65623/FEE0NEM)/2.5) 60 CONTINUE C IF(FEEDNEM.GT.125.) PERNEMA - 0.00022122 • OT NEMAFEO - (PERNEMA * FEEDNEM) * DT C C LIMIT EFFECT OF NEMAS TO 70 ROOT REDUCTION C 1F(NEMAFED.GT.0.70) NEMAFED - 0.70 GO TO 200 100 NEMAFED - 0.0001 200 CONTINUE RETURN END SUBROUTINE SOILTMP 1 5 C C C C c C ....................................................... . •*••*..**•••••*...*• SUBROUTINE SOILTMP ***.*.*•*..***.***•**., c c ..... 10 15 VARIABLES: ...............................................i c* C * C * C • C • C* C * C * JDAY- JULIAN DAY PARAMETER USED TO DISTINGUISH 1ST ITERATION OF SIMULATION (VARIABLES INITIALIZED) FROM SUBSEOUENT ITERATIONS STEMP- SOIL TEMP READ FROM TAPE 1 (RECORDED BIHOURLY) IX * C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SUBROUTINE SOILTMP(JOAY.STEMP.LX) 20 C 25 DIMENSION 5TEMP(13) C C JULIAN DAY COUNTER C JDAY-JDAY ♦ 1 C C INITIALIZE VARIABLES C IF(LX.GT.O) GO TO 100 30 DO 10 1-1,13 READ (1.50) STEMP(I) 35 STEMP(I)10 C CONTINUE GO TO 30 (STEMP(I)-32.) *5./9. CONTINUE STEMPC1)-STEMP(13) DO 20 1-2.13 READ (1.50) STEMP(I) ST E M P (I)- (S T E M P C I)-3 2 .) - S . / 9 . CONTINUE CONTINUE F0RMATCF2.0) RETURN END ro SUBROUTINE MODEL1 t 5 C C C C c C •••••••••••••••••••• C SUBROUTINE M0DEL2 •••***•*••*••• ••••••••••*•••••••****•••***********************«*•«•• c C 10 15 10 20 12 13 25 30 30 31 9 15 40 35 VARIABLES: SUBROUTINE MODEL1(X,AUX.RATE,AXTOT,TOT) DIMENSION X(10).AUX<10) AXTOT-O. DO 10 1-1.10 AUX(I)-X(I) AXTOT-AXTOT+AUXfI) X(I)-0. CONTINUE 00 15 1-1.10 DO 9 J-1.I YMIN-.1-CU-1) YMAX-.1-d IF(RATE~YMIN)9.9.12 IF(RATE-VMAX)19.13.9 IJ-I-«J*1 IU1-1U-1 IF(I O SUBROUTINE MODELS C c S C****••******••*•*****SUBROUTINE MOOEL3* *......... c 10 10 53 15 54 20 55 57 160 SUBROUTINE MOOEL3(X8.AUX.RATE 1.DEC.SURV) DIMENSION AUX(10).XI8(10) AOIN-O. DO 10 1-1.10 XI8(I)«0. 00 160 IM-1,10 IF(10-IM-10.-RATE1)53.160.160 RVAL-RATE1-.1-(10-IM) IF(RVAL-.1)54.55.55 XI8(IM)-RVAL*10.*AUX(IM) 60 TO 57 XI8(1M)*AUX(IM) ADIN-ADIN+XI8(IM)-SURV CONTINUE X8-EXP(-DEC)*X8+ADIN RETURN END ro ro FUNCTION FNL 1 5 10 15 15 20 20 25 25 FNL-DVAL(N+1) GO TO 25 XD-X-XS I-I.O+XO/DX FNL-(XD-FLOAT(I-1)*DX)*(DVAL(1+1)-OVAL(I))/OX+DVAL(I) RETURN END 222 C C .... ....... . c ***•***•••**•••••••• FUNCTION FNL ••*»•**••«*•**•*** C C C OVAL - NAME OF DATA ARRAY C XS - FIRST X VALUE C OX • DELTA FOR INDEPENDENT VARIABLE C N - NO. OF DATA PTS - 1 C X - NAME OF INDEPENDENT VARIABLE C FUNCTION FNL(OVAL.XS.OX.N.X) DIMENSION DVAL(1> IF(X-XS)10,10.S 5 IF(X-XS-N»DX)20.15.15 lO FNL«DVAL(1) GO TO 25 APPENDIX D Efficiency of a Method for Extracting Meloidogyne hapla from Soil INTRODUCnON The purpose of this experim ent was to assess the efficiency of the sugar flotation-centrifugation method used in th e described experim ents to e x tra c t Meloidogyne hapla juveniles from muck soil. E arlier, Kotcon (1979) concluded th a t the equipment used and th e timing of the various stages of th e procedure were im portant variables in determ ining th e number of M. hapla recovered from muck soil. In agreem ent with Seinhorst (1962), he found th a t th e length of th e nem atode in relation to the size of the sieve used in the procedure was also im portant. Kotcon's extraction efficiency for M. hapla was 28.1%, as com pared to 36.7% for Pratylenchus penetrans and 37.4% for Tylenchorhynchus spp., nem atodes considerably larger than M. hapla. M oriarty (1960) found th a t in addition to the sources of experim ental error mentioned above, th e re was considerable variation betw een different observers counting nem atode samples. For this reason I fe lt it im portant to establish a measure of the extraction efficiency for myself and my employees, even though th e technique employed was identical to th a t of Kotcon. A secondary, but perhaps more im portant, objective of the experim ent was to assess th e influence of nem atode density per soil sample on the efficiency of our extraction procedure. METHODS Freshly-hatched M. hapla second-stage juveniles were counted and placed in te st tubes containing ca 10 ml of w ater. The following nem atode densities were isolated (number of replications in parentheses): 223 1(10), 5(10), 25(5), 50(4), 221* 75(4), 100(4), and 200(4). One hundred cm of muck soil, steam ed for eight hours, was placed into each of 51 plastic bags. One tube of nem atode inoculum was added to each bag; the tube was rinsed in ca 5 ml of w ater and also added to each bag; and the soil within the bag was ag itated thoroughly. The sample was then processed by a modified sugar flotation-centrifugation technique (Jenkins, 1964) by an employee who had also processed th e m ajority of th e soil samples for the experim ents described previously. myself using a stereom icroscope. The resulting samples were counted by The bags containing soil and th e tubes containing the processed sample were coded so th a t neither the employee nor myself was aw are of the nem atode density for a specific sample. RESULTS AND DISCUSSION There was a highly significant (P=0.01) linear relation between the initial nem atode density and the number of nem atodes recovered per sample (Figure 50A). According to regression analysis, an average of 34.95 of the nem atodes within a sample were recovered. D ata for th e proportion of nem atodes recovered were transform ed by an arcsine transform ation and regressed on initial nem atode density (Figure 50B). There was no linear relation between the percentage of nem atodes obtained a fte r extraction and initial nematode density, indicating th a t a constant proportion (0.33) of th e nem atodes in each sample were recovered. Inspection of the untransform ed d ata showed th a t the variances of the observations for different nem atode densities were not equal. For this reason, a weighted least squares analysis of the d ata was also perform ed to fit the model: E(y) = bX where: 225 Q Ld o O' oLd Y-0.3545X+0.0919 > R1-0.9528 o l/ J o r Ld ~. O' in in. Ld o A w * m s' Q s' * * *' O n-. < i CM 2 #s \ # Ld Z • n0 1 50 100 150 200 o z NEMATODE DENSITY B o o —| 33.21 Ld O ' Ld in- Ld in.w o - M k k 1— - >* I Ld *K O ' Ld Q_ o 50 100 150 200 NEMATODE DENSITY Figure 50. Number (A) and p ercentage (B) of Meloidogyne hapla recovered per 100 cn>3 s o i l given i n i t i a l d e n s i t i e s o f 1 - 200 nematodes. 226 b= E w.x.y. f t WjX.2 2 2 w. = weighting vaiue 6 /$ • x. = actual nem atode density y. = nem atodes recovered Y, the number of nem atodes recovered from a sample, can be described by a binomial distribution, according to th e following equation: Yi = Pxi xi + error (E(error)=0) (V(error)=kx.) where: Px. = probability of finding a nem atode (constant) E = mean V = variance k = Px.U -Pxp Substituting kx. in the denom inator of the weighting value, Wj, b can be estim ated by: EyJ E x. = y / x According to this analysis, 35.68% (V(b)=0.9107%) of the initial number of M. hapla present in a sample were recovered by the technique employed. The three techniques used to analyze extraction efficiency: (1) regression analysis of counts, (2) regression analysis of transform ed (arcsine) counts, and (3) weighted least squares analysis, produced similar results. It seems then, th a t nem atode density (for densities less than 200 nem atodes per 100 cm soil) was not a factor influencing the results of the experim ents presented here. Many other potential sources of sampling error do exist, however, and may not be consistent betw een different experim ental environm ents. Additional sources of variability in research procedures should be analyzed before an atte m p t is made to directly re la te the data collected in greenhouse experim ents to observations made in th e field. LITERATURE CITED LITERATURE CITED Agrios, G. N. 1978. Plant Pathology. Academic Press, London. 703 pp. Allen, G. E., and 3. E. Bath. 1980. The conceptual and institutional aspects of pest m anagem ent. Bioscience 30:658-664. Atilano, R. A., 3. A. 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