STU‘DES OH WHlTE PWE SEED‘UNG ROOT ROT Thesis {or ‘th Degree of M. S. MICBIGAN STATE U-NE‘VERSITY Jerry Wifiiam Rifle E959 .o.-ofiq.”fifi::..co on... 9' c ...... I LIBRAf-‘zv ‘ ¥ MiChigMI State University STUDIES OK TNITE PINE SEEDLING ROOT RCT For TbJ‘rf‘iY ILL L'U‘a‘. HI FFLE ( An Abstract Submitted to the College of Science and Arts hichigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MA TER OF SCIERCE Department of Botany and Plant Pathology 1959 Approved ?W 6 55734-7 IJJABSfluflT The purpose of this investigation was to determine the causal organism of a root rot disease of white pine nursery seedlings at the U. S. TDorest Service Chittenden Nursery in Uellston, Vichigan. Efforts were made to nemonstrate pathogenicity of a suspected pathogen, to extract and study the population of nematodes from soil and from white pine roots, and to stucy diseased root sections of white pine seedlings microscopically through the preparation of permanent slides of diseased root tissue. Although various microorganisms were isolated on PDA medium from uhite pine root tissue plantings showing various degrees of root rot, Fusarium was the principal microorganism isolated, representing 30.84 percent of all tissue plantings made. Three Fusarium species, namely F. solani, W. oxv- sporum, and F. moniliforme, were used in greenhouse inocu- lation tests. Three series of greenhouse tests were made using Fusarium sgecies to infest the soil in which white pine seedlings and seed were planted. Fusarium was the principal microorganism reiso- lated from the infected plants in the greenhouse. The percentage recovery of Thisarium due solely to the deliber- ate infestation with Pusarium in the first greenhouse series was 52.68 percent. The corresponding figure for the second greenhouse series was 60.26 percent. The mix- .h_~_-.— ‘v cpl.) O“ u’ (an) vNFN‘I ”—0 ul '1" ‘4 ture of the ffiAna'Pusarium isolates (Fflfif)‘was the most pathogenic inoculum used in the greenhouse tests. ‘l'ne nfected white pine seedlings H- percentage recovery from the in this treatment was 09.72 percent. Fusarium oxysporum was the most pathogenic separate inoculum used, for the percentage recovery of this microorganism trom infected plants was 90.80 percent. Population studies of nematodes reveal that many nematodes were present in the soil and plant samples collected throughout the diseased areas of the nursery. Pore nematodes were extracted from the nursery soil than from the woodsplot soil or from the soil of a white pine plantation located near the Chittenden Yursery. Since root rot only occurs in the nursery beds, the evidence suggests that nematodes may play a part in the root rot disease, although their exact role is not known. Nematodes were used to infest the soil in the third greenhouse series. Fusarium and Verticillium species were also used as inoculum in this series, but the latter was not pathogenic. The percentage recovery of ‘usarium in this greenhouse series was 83.55 percent. Histological studies of sections from diseased white pine roots revealed that fungus hyphae were present in the root epidermis and cortex of tw@ diseased.seed- lings. 2" ms TT)T yo , 31V luv .. .L Lin.) UL! By JERRY LILLIAM RIFFLE A THESIS Submitted to the College of Science and Arts Hichigan State University of Agriculture and Applied Science in partial fulfillnent of the requirements for the degree of ’J MKS TE R O F S C IN»? (3 " «7 Department of Botany and Plant Pathology 1959 J. 8;» a' »'. ‘T”/*' '27 C; Z " L- 7 ACKLOHLEDGEHETTS The author is very grateful to Professor I3‘orrest C. Strong and Prof ssor h. C. Strong for suggestions and advice given throughout this investigation, and to Dr. Donald J. deZeeuw and Professor Putnam W. Rotbins for assistance and criticism of this manuscript. The cooperation and assistance which was given by Dr. Ralph L. Anderson of the Lake States Forest Experiment Station, and by Mr. Edward D. Clifford, Superintendent of the Chittenden Forest Nursery, dxring this investigation were greatly appreciated. The author is very grateful to Dr. William C. Snyder, Professor of Plant Pathology at the University of California, vho identified the five Fusarium isolates which were used in this study. Special thanks is extended to Dr. John A. Knierim of the Entomology Department for his advice and for the equipment used in the nematode studies. Lastly, the author wishes to thank all other persons not mentioned above who have offered suggestions and aid pertaining to this problem. These research studies were supported in part ty a grant of $2400 from the U. 8. Forest Service. In addition, certain supjlies and labor funds were furnished by the TT F t. 0. Forest Service. ..‘-' in- .vr-r A» _.. v1 Add .A f“ . I e TABLE OF“ CdIETh'JNTS Page IN‘I‘hOIHCTIUN........................................ 1. Description of the disease.................. 10. REVIEW 0? LITERATURE................................ 17. TITATERIALS19.3517I~'ET??UT;¥'S...............................20. Isolation and identification of micro- organisms................................... 20. Greenhouse inoculation technique............ 21. Hematology.................................. 28. Extracting nematodes from pine root tissue.. 29. Extracting nematodes from soil samples...... 50. icrotechnique studies”.................... 55. 1‘1“" ‘1’. ‘T' ‘\Tf{fi- -' f-\ 'fi‘ {r- x‘rj- :l-ArfivlTN mo ,7 DJLfLFKlLIALJ&\J.Hl-J UhLli M.l‘l-) leu-JL-‘A. ....................... 0:5. Isolation studies... 0 O O O O O O O O O O O U U 0 InOCUlation StUdieSooooooooooooo000000000000 360 NematOde StudiGSOOQooooooooooooooooooooooooo 57. DISCUSSION AND CUNCL”SIONS...-ooooooo.......oooooooo 68. SWMMAhYoooooooooooooo.oo000.000.00.000.000.000.0000. 75. LITbimTY-II‘E CI'IEI)OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 77. LIQT OF TABLES Page I. Frequency of microorganisms isolated from white pine nursery seedlings showing various fiearees Of POOt rOtoooooooooooooooooo 380 II. Source of Pusarium isolates used in inOCUlatlon tests............................ 40. III. Frequency of microorganisms isolated from 1-0 white pine nursery seedlings showing various degrees Of POOt rOtoooooooooooooooooo 410 IV. Frequency of microorganisms isolated from 2-0 white pine nursery seedlirgs showing various degrees of root rot.................. 42. V. Frequency of microorganisms isolated from 2-1 white pine nursery seedlings showing various degrees of root rot.................. 45. VI. Frequency of microorganisms isolated from 2-2 white pine nursery seedlings showing VHPiOUS degrees 0? TUOt POt-ooooooooooooooooo 44. VII. Frequency of microorganisms isolated from ‘white pine noodsplot seedlings............... 51. VIII. Frequency of microorganisms isolated from white pine woodsplot seedlings tranSJIanted into the nursery transplant deSooooooooooooo 53. IX. Frequency of microorganisms reisoluted from white pine seedlings grown in soil in the -irst and second greenhouse series........... 55. X. Frequency of microorganisms reisolated from white pine seedlings grown in soil in the first greenhouse series...................... 57. XI. Frequency of microorganisms reisolated from white pine seedlings grown in soil in the second greenhouse series..................... 59. XII. Frequency of microorganisms reisolated from white pine seedlings grown in soil in the third greenhouse SCPIGS...................... 62. XIII. Genera of nematodes associated with white pine seedlings in the Chittenden Nursery in1950.00.00.00000000OOOOOOOOOOOOCOOOOOOOOOO 64. XIV o XV . XVI o C Page XIV. Species of nematodes associated with white pine seedlings and nursery soil in the Chittenden Fursery in 1950............ 65. XV. Frequency of nematodes extracted Prom soil samples collected from nursery plots, the woodsplot, and a white pine plantation near the Chittenden Nursery.................. 66. XVI. Frequency of nematodes extracted from the roots of diseased white pine seedlings collected at various locations in the Chittemlen Nursery-00.0.0000000000000000oocoo 670 10. 11. 12. 15. L IS T L7 F FIGTTRTCS Graph of frequency of microorganisms isolated from white pine nursery seedlings showing various degrees of root rot......... Graph of Fusarium frequency on HinPrent age StOCkooo00000000000000.0000... Graph of non-sporulators frequency on different age StOCkooooeuocooooooooo Graph of Trichoderma frequency on different age stock................. Graph of Penicillium frequency on different age stock................. Graph of Rhizopus frequency on different age StOCkooooooooooooooooo Graph of Bacteria frequency on different age StOCkooooooooooooooooo Graph of fre uency of microor*anisms - J 5 isolated from white pine woodsplot seedlings Graph of frequency of microorganisms isolated from white pine woodsplot seedlings transulanted into nursery transplant Graph of frequency of microorganisms reisolated from white pine seedlings in soil in the first and second beds... grown greenhouse serieS..........................o Graph of frequency of microorganisms reisolated from white pine seedlings grown in soil in the first greenhouse series...... Graph of frequency of microorganisms reisolated from white pine seedlings grown in soil in the second greenhouse series..... Graph of frequency of microorganisms reisolated from white pine seedlings grown in soil in the third greenhouse series...... 49. 50. 52. 56. CO. '12 Go. I V. LIST 0? PLATES Page iortion of woodsplot area where no rOOt POt OCCUPSOOOOOOOOOUIOIOOOOOOOOOOOOOOOOOO 4. Nursery control plots showing 2-0 white ° ‘1! . O p_-ne SCG(.].L.LI1&JSQ0000000000000000000000000.0000. 1‘40 Hursery plot containing 2-0 white pine seedlings in which the soil was treated with formaldehyde (fourty percent commercial at the rate 1.5 ounces per square foot........ 3. I V Nursery plot containing 2-0 white pine seedlings in which the soil was fumigated With methyl bromide at the rate of two IDOImdS 1381" 100 SQ‘kiaPe feet.00000000000000.0000 34. Hoot rot symptoms on a 2-2 white .ine transplantoooooo00000000000.000.000.900.000... 100 Stages of root rot symptoms on 2-2 white pine transplantSoooooooooo00000000000000.0000. 16¢ Diagrammatic sketch of inverted bottle used to collect nematodes from soil and plant SampleSoo0.00000000000000000000000000000 52. Growth on FDA medium of Fusarium reisolated from white pine seedlings of the second {51169111101188 serieSOOOOOOO0.00000000000000000000 610 Fore stri fore cans tain Of'o INTRODUCTION From a commercial standpoint, white pine, Einus strobus L., is considered the most valuable species of this type in the Lakes States Region (22). It has the highest board feet value of any conifer species grown in this region. At the Chittenden Nursery less than ten percent of the total plantings at the present time are white pine, the balance consisting of red pine, flags resinosa Ait. Approximately 250,000 white pine trans- plants are transplanted yearly on the Manistee National Forest. One of the serious limiting factors that re- stricts an increased number of white pine outplantings in forestation areas is the Miite pine blister rust disease, caused by Cronartium ribicola Fisch. The cost of main- taining'fiibgs free areas prohibits an increased percentage of outplantings of white pine. .Bihfii species, currents and gooseberries, are the alternate hosts of white pine blister rust fungus. Still another limiting factor that restricts the outplanting of white pine is the possibility of attacks by the white pine weevil, Pissodes strobi Boh., which deforms the trees and seriously interferes with their growth rate. These factors, and others, explain why less than ten percent of the total production of seedlings at the present time at the Chittenden Nursery are white pine. Since 1954, a serious loss of white pine seedlings has occurred in the seedbeds and the transplant beds of the Chittenden Nursery, Plate II. Death of the l. seedlings follows what appears to be a gradual dying of the roots characteristic of the root rot disease of the white pine seedlings. This disease condition does not affect red pine seedlings grown in the same seedbeds with white pine seedlings nor does the disease continue to kill white pine after they are transplanted into a plantation. Eastern white pine is the main species affected by the disease. Although the disease has also been found on white spruce, Eigeg glauca (Moench) Voss, and black spruce, Picea mariana (Mill.) B.S.P., it causes very little loss to these two species. A planting was made in a newly cleared area in the oak woods adjacent to the nursery in order to determine if a root rot pathogen capable of affecting white pine seedlings was present, Plate I. This area near the south border of the nursery was cleared in the fall of 1954. The seedbed, four feet wide and twenty-five feet long, was sown with white pine seed in the spring of 1955. In the fall of 1956, a crop of seedlings was removed and transplanted into the main nursery to see if they would be affected with the root rot disease. The woodsplot area was immediately resown with white pine seed. No case of root rot has originated from the woodsplot since its establishment. Lack of root rot in this area may be due to a different composition of microflora coupled with the fact that it is a virgin area as far as a nursery geedb PemO‘J nu? S E den the seedbed is concerned. The second crop of seedlings was removed in the fall of 1958 and transplanted into the nursery as before in order to determine their suscepti- bility to the root rot pathogen. The area was to be immediately resown with white pine seed. It is tenta- tively planned to continue growing white pine seedlings on the area to determine how long it will remain free of the root rot disease. The fact that the root rot disease has not developed in the woodsplot area supports the other observations made on surveys of outplanted white pine plantations. Very few white pine succumb from root rot after they are outplanted on planting sites within the forest. A review of the production records of the Chitten- den Nursery since its establishment in 1934 shows that there have been consistent losses in the white pine beds. At first these losses were attributed to white grubs (2). In 1943, it was found that white pine were dying from some other cause than white grubs. In 1948, the root rot disease was recognized in the 2-1 and 2-2 white pine transplants, and a large number of trees were lost in both ages of stock (2). The possibility of white grub injury was investigated, but there was no evidence of such injury on the majority of the transplants that had died. The root rot disease was extremely serious in 1949. Nost of the 1-0 white pine seedlings were killed and heavy losses occurred in the 2-0, 2-1, and 2-2 beds (2). In 3. PLATE I q a r.‘ a t . .uo _ l Portion of the woodsplot area where no root rot occurs. 4. 1950, 250,000 white pine seed were sown and from this thirty-six percent were available for transplanting and twenty percent for shipping. It was learned that the W.W. Ashe Nursery in Mississippi had suffered heavy losses of their southern pines from a root rot disease. It was found that if the nematodes present at the nursery were controlled, the root rot disease failed to develop. Samples of 2-1 and 2-2 white pine from the Chittenden Nursery were sent to Dr. G. Steiner, principal nematologist at the Plant Industry Station at Beltsville, Maryland, in 1949. He found several nematodes, most of which were different from those at the Ashe Nursery. In a letter to Mr. E.D. Clifford, Superintendent of the Chittenden Nursery, dated August 5, 1949, Dr. Steiner said that in general the nematode complex was quite similar to the one seen at the Ashe Nursery. Additional samples of stock were sent to Dr. G. Steiner in 1950, and he found the same nematodes in these samples as in the past and a few additional ones- (Table XIII). The genera found were Alphelenchus, Alphelenchoides, Acrobeloides, Ditylenchus, Dorylaimus, Metaphelenchus, Metarhabditis, Tylenchus, Plectus, Panagrolaimus, Acrobeles, Diplogaster, and Seinura. The Aphelenchus and Panagrolaimus genera were the most numerous and both are considered to be decay nematodes (2). Dr. Gerald Thorne, a noted nematologist, visited 5. the Chittenden Nursery in 1950 with Dr. Curtis E. Dieter, of the Dow Chemical Company, and collected soil and plant samples. They found several nematodes in the samples, of which the following are able to cause injury to plants: Leptonchus granulosus, Tylenchorhynchus dubius, Aphelen- choides sp., Ditylenchus sp., and Tylencholaimus proximus (Table XIV) (2). Dr. Berch Henry, from the Ashe Nursery, visited the Chittenden Nursery in the spring of 1950. He and others established experiments employing the use of soil fumigants and insecticides in an attempt to control the root rot disease. The disease was not generally serious in 1950, and results of the experiments showed that the fungicide and insecticide plots failed to give any indi- cation of control of the disease (2). During 1951, Dr. Henry grew red pine and white pine in infected areas at the Ashe Nursery. Both species proved to be susceptible to the Ashe root rot, while only the white pine seemed to be susceptible to the root rot found at the Chittenden Nursery. Since 1952 various experiments have been carried out in an effort to control the root rot disease. Soil fumigants have been tested on a large scale, and some tests have been made with fungicides, insecticides, and fertilizers. Results of tests as of 1956 by the Lakes States Forest Experiment Station involving over 500,000 white pine seedlings indicate the following (1): O. 1. Methyl bromidea applied at the rate of two pounds per 100 square feet, supplemented with a Parzate (zinc ethylene bisdithiocarbamate) foliage spray, has increased production of seedlings by as much as 150 percent over that of untreated beds. Methyl bromide alone and methyl bromide plus Fermate (ferric dimethyl dithiocarbamate) sprays also show large increases in production, Plate IV. 2. Formaldehyde (forty percent commercial) applied at 1.5 ounces per square foot, or about three times the dosage generally recommended for damping-off control, has given excellent control of the root rot disease, equal to or better than methyl bromide, Plate III. 5. The use of methyl bromide and formaldehyde has resulted in heavier seedlings even though there was a denser stand in these beds. Thus the white pine root rot problem at the Chittenden Nursery has received some attention for several years. Uith the cooperative help of the nursery staff, the Dow Chemical Company, the pathologists and nematolo- gists of the Plant Industry Station, and the Lakes States Forest Experiment Station disease research personnel, a Methyl bromide (ninety-eight percent methyl bromide plus two percent chloropicrin) contributed by the Dow Chemical Company, Midland, Michigan. 4 several experiments have been conducted in an effort to find satisfactory control measures. At the present time, methyl bromide applied at the rate of 1.5 pounds per 100 square feet, and formaldehyde, applied at the rate of 1.5 ounces per square foot, are the most effective of the various chemicals used to control the disease. Before fumigation of the soil with methyl bromide was adopted, at least ninety percent of the white pine stock going out of the nursery was affected with root rot ranging in severity of infection from light to heavy. White pine seedlings exhibiting extreme infection were culled. In the last two years, through the use of methyl bromide, this percentage has been greatly reduced. It was estimated by Mr. Clifford that less than five percent of the white pine stock which left the nursery in the fall of 1958 was affected with root rot. In 1954 a survey of three white pine plantations which were planted in 1952 revealed that the average loss of white pine was ten percent. Two percent was entirely accountable to root rot, and the other eight percent was due to transplanting. It was found some time later that death of white pine due entirely to root rot does not occur after two years in the field, although death may result from a combination of root rot and transplanting injury. After the third year in the field, death due to the combination of root rot and transplanting does not 000111" o The above observations offer an interesting comparison when related to outplantings with diseased stock at the Ashe Nursery by Dr. Perch Henry. Data in a letter from Dr. Henry to Dr. Curtis May, principal nema- tologist of the Bureau of Plant Industry, dated September 12, 1949 indicated that of the outplantings made from 1947 experimental plots at the Ashe Nursery, the first year survival of "plantable" slash pine seedlings, Pinus elliottii Bngelm, was not adversely affected by the root rot. The survival of longleaf pine, Pinus palustris Mill., after one year in the field, ranged from fourty- four to ninety-two percent. Dr. Henry indicated that Dowfume W—40, applied at the rate of thirty—two gallons per acre, 2.5 to 5 weeks before spring sowing, has given satisfactory root rot control of slash and longleaf pine seedlings in the Ashe Nursery for two consecutive years. It has become apparent that the cause of the root rot disease at the Chittenden Nursery must be determined so that better control methods can he applied. Studies were initiated in June 1957 to determine the actual cause of the root rot disease, and they were continued through August 1958. These studies were divided into three phases, which are the isolation and reisolation of micro- organisms from diseased white pine seedlings, the ex- traction of nematodes from plant and soil samples for population studies, and the histological studies of sections of diseased white pine seedlings. 9. Description of the disease Root rot occurs on white pine nursery stock of all ages, from 1-0 seedlings to 2—2 transplants, throughout the nursery. It is common to find healthy and affected trees in all stages of decline intermixed in the white pine beds of the nursery. The aboveground symptoms of the root rot are ctlorosis and stunting of the foliage, and a stunting of the tree itself. The stunting and chlorosis of the foliage appears to progress from the top of the seedling downward. The color of the foliage changes from a dark bluish green to a color ranging from a pale green to a light yellow. The color change starts on the extreme tips of the needles and advances toward their base. Later the needles droop, dry up, and turn brownish red, from the tip downward. The seedlings die in an upright position after the bark and cambium are killed by the pathogen, Plate VI. The small rootlets rot first, followed by the lower portion of the primary root, Plate V. Some of the lateral roots on the healthy appearing trees have root rot symptoms. The cortex is sloughed off from the affected lateral roots as they become well decomposed with the advance ofthe disease. Before the cortex is sloughed off, the epidermis turns from a light tan to a black color. This color change is probably due to the breaking down of the root tissue hy the pathogen. Free hand cross sections 10. made at points where the epidermis is blackened reveal a light brownish discoloration of the woody tissue. The root rot progresses from the young tissues of the root toward the older tissues, and proliferation occurs above the rotted portions of the lateral roots. Later, portions of the lateral roots and primary roots completely slough off leaving only a small part of the root system. Trees at this stage of decline are sustained by a few lateral roots restricted to the upper few inches of the soil. ll. Nursery control plots showing 2-0 white pine seedlings.a a Black and white photograph courtesy of Dr. Ralph L. Anderson of the Lakes States Forest Experiment Station. PLATE II 12. PLATE III Nursery plot containing 2-0 white pine seedlings in which the soil was treated with formaldehyde (forty percent commercial) at the rate 1.5 ounces per square foot. 13. PLATE IV Nursery plot containing 2-0 white pine seedlings in which the soil was fumigated with methyl bromide at the rate of two pounds per 100 square feet.a’b a The darkened areas in the foreground of the colored photo are shadows. Black and white photograph courtesy of Dr. Ralph L. Anderson of the Lakes States Forest Experiment Station. PLATE IV 14. PLATE V Root rot symptoms on a 2-2 white pine transplant. 15. . v 111‘ _ PLATE VI 4 ”--_-~—-‘ ._—__ _‘ _ Stages of root rot symptoms on 2-2 white pine transplants. Healthy transplant on left, diseased transplant in middle, and dead transplant on right.8 Courtesy of Dr. Ralph L. Anderson of the Lakes States Forest Experiment Station. 16. REVIEW OF LITERATURE Many early workers have found that Fusarium species were pathogenic on pine seedlings. Inoculation experiments by Spaulding (17) established that some strains of Fusarium were parasitic and caused damping-off of pine seedlings. Eusarium moniliforme Sheldon was one of the more virulent species. A few years later Gifford (5) repeatedly isolated a species of Fusarium capable of causing damping-off and root rot from the roots of two-year-old white pine seed- lings. The findings of Hartley et al (8) indicated that Fusarium solani (hart.) Appel & Wr. was a weak parasite on jack pine, Pinus banksiana Lamb., while the most destruc- tive of the species of Fusarium tested wale. moniliforme. Variable results were obtained by Rathbun (15) using strains of Fusarium species involved in a root rot of jack pine and red pine, root rot being more severe in the Fusarium inoculations than in the controls. Eliason (5) found that white pine and white spruce were intermediate in susceptibility to a Fusarium root rot. In this study there was a definite relation shown of buckwheat residue to root rot. The root rot was more severe in the nursery areas which were successively sown for three or more years with buckwheat as a cover crop. Tint (18) demonstrated that resistance to invasion of Fusarium oxysporum Schlect. in seedlings of red pine increased directly with increasing age of the seedlings. Various other workers have reported 17. damping-off of pine seedlings caused ty Tusarium (10, 19, 21). A Fusarium has been found in what appears to be a nematode-fungus complex in the Ashe Forest Nursery in Mississippi. Longleaf pine, slash pine, and loblolly pine, Iinus taeda L., were susceptible to root rot (l4). In plots most severely affected, fifty percent of the loblolly pine, ninety percent of the spring sown longleaf pine, and ninety-five percent of the slash pine died. It appeared, from the survival in the nursery and the survival in the field, that the root rot affected spring sown longleaf p'ne most severely, loblolly pine next, and slash pine the least. Fusarium, two other species of fungi, and representatives of at least ten genera of nematodes were found. Strong evidence indicated that the cause of the disease was a nematode-fungus complex, since application of the nematocidal fumigant ethylene dibromide to the seedbeds at the rate of twenty—four gallons per acre of a twenty percent by volume solution two to three weeks prior to spring seeding gave excellent root rot control. To phytotoxic effects resulted from this rate of application (9). Nematologists have established the fact that para- sitic nematodes are associated with the roots of all the major southern pine species (7). A parasitic nematode identified as Criconemoides rusticum (Miceletzky Taylor was associated with the deterioration of the fine roots and mycorrhiza of one-year-old potted shortleaf pine seed- lings, Pinus echinata Mill. (l2). Hepper (11) made a survey of the plant parasitic nematodes in the soils of thirty-five southern forest nurseries and found that Neloidodera and Tylenchorhynchus were the only nematode genera found to be directly associated with seedling injury. Tylenchus was frequently associated with.root rots but was not considered responsible for this injury. Root-knot nematodes have been found in connection with Fusarium wilt of mimosa, Albizzia julibrissin Durazz (G). More wilting of mimosa occurred when the soil was 0 infested with Tusarium oxysporum l. perniciosum (Tepting) Tools and teloidogyne incognita (Kofoid & White) Chitwood or N. javanica (Treub) Chitwood in combination than in soil infested with Eusarium alone. The nematodes alone reduced the size of the mimosa seedlings. NATERIALS [£37 7.133“??st Isolation and identification of ricroorganisms The isolation and identification of microorganisms from white pine seedlings showing symptoms of the root rot disease was initiated in June 1957 at the U.S. Forest Service Chittenden Nursery at Wellston, Vichigan. Working on the premise that a soil inhabiting fungus might be the cause of the root rot, tissue plantings were made from the roots of white pine seedlings in various stages of decline. The nursery stock selected for tissue planting was brought into the laboratory located on the nursery grounds. The roots were thoroughly washed in flowing tap water in order to remove excess soil and other foreign matter. Roots showing early stages of browning were selected and cut into sections one-quarter to one-half inch in length. Seventy percent ethyl alcohol was used as a surface sterilizing agent. Each root section was immersed in this solution for thirty to sixty seconds and then aseptically transferred into a bath of sterile distilled water for one minute before planting aseptically with forceps on sterile medium in a Petri plate. Five root sections were planted in each plate. as principal medium used was potato dextrose agar (PDA) acidified with twenty-five percent lactic acid. The tissue planted plates were then labeled, wrapped in wax paper to prevent aerial contamination, and incubated at room temperature. These plates were observed 20. macroscopically three to six days after plating. The colonies growing from each tissue planting were identified to genus as far as possible by examining them on microscope slides under a compound microscope. The fungi that did not sporulate were transferred to FDA slants and attempts were made to identify them approxi- mately one to two weeks later. All transfers were made from the outer edge of the young, actively growing colo- nies. After identification, the cultures were transferred to FDA slants and maintained in the refrigerator. The frequency of microorganisms isolated in this study is shown in Table 1. Greenhouse inoculation techniqge Since Fusarium species appeared consistently and predominantly throughout the isolation studies conducted during the summer of 1957 (Table I), it seemed desirable to test the pathogenicity of these organisms. Five Fusarium isolates, numbers 35, 42, 46, 49, and 62 were selected at random from the numerous collection of pure culture isolates of 1957. The source of these isolates i -hown in Table II. The five isolates were grown on (I) C) two different media. One medium consisted of cornmeal plus vermiculite and the other was a synthetic liquid medium. The cornmeal plus vermiculite medium was prepared by mix ng 150 ml. (by volume) of yellow cornmeal in a one 21. liter Erlenmeyer flask with 150 ml. each of water and vermiculite. These materials were stirred with a glass rod until thoroughly mixed. Six of the flasks thus pre- pared were then autoclaved for twenty minutes at fifteen pounds pressure, allowed to cool, and each flask was planted with one of the selected Fusarium pure culture isolates. One flask was left unplanted for use as control inoculum. The synthetic liquid medium was prepared by adding the following material to one liter of distilled water in a sterile Erlenmeyer flask: 7.2 grams sucrose, 5.6 grams glucose, 1.25 grams MgSO4, 2.72 grams KH2P04, and 2.02 grams KN03 (16). I Ten liter flasks, each containing 200 ml. of me- dium, were prepared. The flasks were autoclaved for twenty minutes at fifteen pounds pressure and allowed to cool. Five flasks were then planted each with one of the selected Fusarium pure culture isolates. five flasks were left unplanted, three of them for control inoculum and two for use in suspending the mycelial mats when they were macer- ated in the Waring blender. The inoculum used for the first greenhouse series was allowed to grow on the medium for eighteen days. The pure sand which was to be infested with the inoculum was obtained from a sand pit located approximately five miles east of the Chittenden Nursery. The sand was collected in metal containers at a depth of thirty-five 232. feet telow the ground leve_, and it was considered free of organic matter and reasonably sterile. hanger of intro- ducing the root rot pathogen from this source was con- sidered to be negligible because of the very low organic content of the sand. The sand was brought to East Lansing and placed in newly constructed flats in the Plan Science Greenhouse. The inoculum, consisting of the contents of one Erlenmeyer liter flask. was dumped on the sand surface, broken into small pieces, and mixed into the sand by hand. After thoroughly mixing the inoculum into the sand, the sand surface was leveled and a new metal divider was * aced to A H- in the flat crosswise to separate t Into two equal parts. One-half of the flat was planted with one hundred white pine seeds from the 1956 crop, fifty seeds per row. The 7e flat was planted with A. J- 7 ”P o ther ,zal f 61 t .1 .0 . -, ‘ .\- 1 ‘3 . 1.9L a Mcwlspiut U) C) L }._J _.J (D 0 c1- 0 ,1; p C'- r+ - ~71 3 a f‘:( "'5 ‘4; in. he Chittenden Nursery and trans- 11 spia;urnn moss ill plastdxz he s. w ported to East Lansing q These seedlin's and been planted in the fall of 1956 in a acent to the south side of the nursery proper. (.41 toodspl ot ac. No root rot has occurred in tiis plot, but some of the seedlings from it that have been planted in the nursery developed root rot. These woodsplot seedlings were transplanted into greenhouse flats as quickly as possible. Fifty seedlings, five rows of ten each, were planted in each flat. The flats were put in four inch deep metal pans and watered from below. The soil in the rest of the flats receiving {\I‘. ()1 o inoculum grown on cornmeal vermiculite was infested in the same manner. The check flats were prepared first in order to avoid contamination from the other flats. The synthetic liquid medium was decanted from the mycelial mats of the five Fusarium isolates into a liter flask and was used for one treatment. Unplanted synthetic liquid medium served as a control treatment. The mycelial mats of the five Fusarium isolates, which were grown on the synthetic liquid medium for eighteen days, were suspended in 400 ml. of unplanted synthetic medium in a Waring blender. The mycelial mats were macerated for one minute. After the greenhouse flats were p anted with one hundred white pine seed and fifty woodsplot seedlings, the mycelial fragments and spores suspended in the liquid medium were sprayed on the surface of the sand in the flat with a rubber bulb sprayer. The same inoculation pro- cedure was used for the 600 ml. of unplanted liquid medium control and for the decanted liquid from the mycelial mats. The first greenhouse series consisted of ten flats as follows: 1. Unplanted liquid medium check (PLM) 2. Unplanted cornmeal vermiculite medium (CCV) 5. Control - no medium or inoculum added (CNN) 4. Decanted liquid from the five Wusarium mycelial mats (LFM) 5. Five Fusarium mycelial mats macerated in sterile liquid medium (FMM) 24. 6. Tusarium isolate 33 - Cornmeal vermiculite medium 7. Fusarium isolate 42 - Cornmeal vermiculite medium ,4 11 Ho c+ CD 8. Fusarium isolate 46 - Cornmeal vermie medium 9. Fusarium isolate 49 - Cornmeal vermiculite medium 10. Fusarium isolate 62 - Cornmeal vermiculite medium Periodic counts were made on the number of seed germinating and on the number of seedlings dying. Uhen the seedlings looked as though they were starting to de- cline or become discolored, they were removed and used in attempts to reisolate the original Fusarium infested into the soil. Some of the seedlings were collected for histo- logical studies. The second greenhouse series was begun in December 1957. The same procedures and inocula previously employed were used with the following exceptions: l. The inoculum was allowed to grow on the two types of media for forty-three days. 2. The liquid from the five Fusarium mycelial mats was discarded. 3. The same type of sand was used except that it was steam sterilized for two hours. 4' The white pine seeds were surface steri- lized in 1:5000 H3612 for five minutes and rinsed in flowing tap water for twenty minutes. The flats were watered from above. Even though the white pine seed were sub- jected to the standard cold treatment, they germinated very poorly in the flats of this second greenhouse series. Therefore, an additional one hundred seeds of the 1957 crop were surface sterilized as above and planted in these flats. The second creenhouse series consisted of ten U flats as follows: 1. 2. Unplanted liquid medium check (PLM) Unplanted cornmeal vermiculite medium (CCV) Control - no medium or inoculum added (CNN) (‘1 Jontrol - no medium or inoculum added (CNM) 7-1 rive Fusarium mycelial mats macerated in sterile liquid medium (FMM) Cornmeal vermiculite m I Fusarium i olate 33 medium Fusarium isolate 42 Cornmeal vermiculite medium Cornmeal vermiculite Fusarium isolate 46 medium PC 0) O 9. Fusarium isolate 49 - Cornmeal vermiculite medium 10. Fusarium isolate 62 - Cornmeal vermiculite medium The flats of the first and second greenhouse series were fertilized once with Folium, a completely soluble fertilizer. Two gallons made at the recommended rate of one ounce per gallon were used. Also one appli- cation of the insecticide dieldrin was applied. A third greenhouse series, using artificially infested soil in metal cans, was begun in May 1958 for the purpose of demonstrating a possible nematode-fungus com- plex. The soil used in this series was obtained from the root rot infested white pine beds at the Chittenden Iursery. The soil was put in no. 10 metal cans and steam sterilized for one and one-half hours. Three Fusarium isolates, namely numbers 55, 42, and 62, were grown on cornmeal vermiculite for forty-four days. The series consisted of the following treatments: 1. Unplanted cornmeal vermiculite medium (CCV) O I \ . Control - no medium or inoculum added (CNN) 5. Nematodes only 4. The three Fusarium isolates only 5. Nematodes plus the three Vusarium isolates 6. The Verticillium isolate only Each treatment was replicated four times. Fusariuw 27. was mixed into the soil by pouring enough soil to fill eight no. 10 metal cans into a fiberglass container and mixing uniformly into this soil the three Fusarium iso- lates. The Verticillium isolate was mixed into the soil in the same manner. After the fungus inoculum—soil mix- ture was placed in the no. 10 cans, ten woodsplot seed- lings were planted in the soil of each can. 0 a O C In the tests involVing nematodes , five times the normal load of nematodes present in the soil was added to each can. Care was taken to see that the soil in these cans remained moist so that the nematodes would not die from dessication. Hematology Samples of affected white pine stock and soil samples were collected from infested areas for the purpose of extracting nematodes. The root systems of the affected plants were carefully lifted from the soil with a spade. Most of the soil samples Jere collected with a soil stab auger from areas representing different age classes of the white pine nursery stock. Soil samples were collected from the upper layer of the soil in which the root systems (bf the white pine seedlings were growing. Each sample rionsisted of approximately one quart of soil. The soil Estab auger was wiped with a clean cloth after taking each See pages 29-55 ftm'extraction of nematodes. 28. sample in order to reduce contamination among the samples. The soil auger was sunk into the soil four times per plot, and a total of three plots represented one sample. The plant and soil samples were collected in polyethylene bags, and care was taken to see that they were kept in a moist condition and were not allowed to become overheated. Extracting nematodes from pine root tissue The affected plants were taken to Dr. Knierim's hematology laboratory where the root systems were care- fully vashed in flowing tap water to remove the excess soil adhering to the roots. The roots were then cut into pieces approximately one-half inch long. The sections were put into a Waring blender with a small amount of water and macerated for two minutes. The sample was washed from the blender with a small stream of water into the inner plastic basket of the two cloth bottomed plastic baskets supported in the inverted bottle which was half full of water, Plate VII. This arrangement allows the nematodes to settle out into the culture tube which is attached to the neck of the inverted bottle by a short piece of rubber hose. The cloth bottoms of the plastic baskets stop the residue from enter- ing the culture tu e. The sample remained in the inverted bottle for three days, during which time the culture tube containing the nematodes was changed daily. After the nematodes were collected in the culture tube, water was drawn off until one-half ml. remained, and 29. 013* LU a D 7 u ,u\ «C 0.1. 74 M.“ .1 a 94 Cu the tube was then immersed in a hot water bath at 1450F. for sixty seconds to kill the nematodes. A fixing solu- tion was then added to the culture tubes to preserve the nematodes for later study. The fixing solution was made up as follows: eight parts water, one part formaldehyde (56.8%), and enough glacial acetic acid so that the fixa— tive solution would contain two percent acetic acid. f‘ Extracting nematodes rem soil samples The soil samples which were collected from in- LL festea areas were taken to the nematology laboratory. A sample was placed into a plastic container containing enough water to thoroughly cover the soil sample. The sample was then stirred and worked with the fingers until all the soil lumps were pulverized and the nematodes were suspended in the water. The soil was allowed to settle momentarily. The water was then decanted from the soil through a screen having openings of 140 microns into another plastic container. More water was added to the soil, and the sample was swirled again. Then the water containing the nematodes from the soil sample was poured through the screen into the other plastic container. This procedure of aiding water to the soil, stirring the soil, and pouring the water containing the suspended nematodes through a screen into the other container was repeated nine times for each sample. The water in the other plas- tic container which contained tho nematodes in suspension 50. was then poured through another screen having an opening of forty microns. This screen allowed water to pass through but retained the nematodes. The nematodes were washed from the screen into a container and then into a .3 container in the inverted bottle, ilate VII. This washin" containing the nematodes remained in the inverted bottle for 24-72 hours, at which time the culture tube at the base of the inverted bottle was removed. The nematodes in the culture tube were taken to the greenhouse, and the soil containing white pine stock was now indested with nematodes ty making two openings in tee soil with a knife and pouring the nematodes into these openings. For the purpose of nematode population studies, soil and plant samples were collected from the nursery. A total of seventy-four samples were collected, eighteen of which were plant samples and fifty-six of which were soil samples. The soil samples were processed by placing each salple into a plastic container and mixing it thoroughly by hand. A 250 ml. soil sample from each collection was placed into the inverted bottle without processing it tin ough the screens, Plate VII. It was not considered necessary to process the soil samples through the series of screens for the population studies alone. The culture tube at the base of the inverted bottle was changed every twenty-four hOlPS during a three day period. The samples were replicated three times. The container used to scoop up the soil was washed in flowing tap water 31. -PLATE VII inner plastic container outer plastic container ater level sample containing nematodes ewes: 223251: .-.W.€::-‘.. first cloth bottom second cloth bottom inverted bottle funnel rubber hose connection culture tube contain- ing nematodes from the sample Diagrammatic sketch of inverted bottle used to collect nematodes from soil and plant samples. after handling each sample in order to reduce factors which would give inaccurate population counts. The plant samples were processed in the same manner as described preViously. Ficrotechnique studies In order to determine early progress of the patho— gen in the host, lOG outwardly healthy plants were col- lected from the inoculated and control flats in the greenhouse. Newly germinated seedlings as well as woods- plot seedl’ngs were collected. Disease? and healthy white pine seedlings were also gathered from the Chittenden Nursery. One hundred and thirty-seven sections one-half inch long were cut from these specimens. The sections were then killed and fixed in FAA (15). The sections remained in this solution for a minimum time of fifty hours, and then t'iey were dehydrated with ethyl alcohol and tertiary butyl alcohol by passing them through a series of changes over a period of two days, the last change being pure tertiary butyl alcohol. The root sections were then transferred to a mixture of equal parts of paraffin oil and butyl alcohol for three hours. 17ollow- ing this, a vial three-fourths full of paraffin, 560- $03. melting point, was cooled to the solidification point. The contents of the vial containing root sections in paraffin oil and tertiary butyl alcohol was poured on top 35. . L, :n' c! t1 0 of the solidified paraffin. This open vial was then placed in the embedding oven for three hours. The solu- tion was then replaced with three changes of fresh paraf- fin over a period of twenty-four hours, the final change being embedding tissuemat. The solution was left in the oven for three hours, after which the sections were cast into blocks. The embedded sections were then mounted on wooden blocks, and serial sections were cut at twelve microns with a rotary microtome. The sections were then mounted with Haupt's adhesive (13). Two stains, Stoughton's Thionin and Orange G, and Pianese III B stain were used for staining (13). Permanent mounts were made with the synthetic resin "clarite". EXPEI‘ZITTEEI‘ITAL DATA AND RESULTS Isolation studies The isolation of microorganisms from white pine nursery stock was carried out during the summer months of 1957 and 1958. Five tissue plantings from the root sys- tems of white pine seedlings exhibiting various degrees of root rot were planted in each Petri plate. The percentage occurrence of each microorganism was calculated and listed in Table I and Figure I. Table I lists the microorganisms isolated from the white pine nursery stock, the number of times each microorganism was isolated, and its percentage occurrence in the total tissue plantings. The non-sporu- lators represent all the microorganisms which failed to sporulate on the medium used for the isolation studies. The results of the isolation studies made through- out this investigation were arranged according to age of white pine nursery stock, and they appear in Tables III- VI and Figures 2-7. These tables show the microorganisms isolated from the total tissue plantings made in this study as to 1-0, 2-0, 2-1, and 2-2 nursery stock. The first number of these values above refers to the number of years a seedling was grown in the seedbed, while the second number refers to the number of years it was grown in the tranSplant bed. White pine seedlings grown in the root rot free woodsplot near the south border of the nursery also were collected for isolation studies. The results of these 55. isolations are shown in Table VII and Figure 8. Table VIII and Figure 9 give the isolation results from nursery transplant bed seedlings that had previously had two years growth in the woodsplot. Tissue plantings were made on unacidified PDA medium to determine the presence on secondary organisms and contaminants. Many colonies of bacteria and other microorganisms appeared around several of these tissue plantings, and in some instances the bacteria covered a considerable portion of the Petri plate. In another series of isolations made, tissue sections were planted directly onto the medium without surface disinfection. It was found that many of the lower fungi .ppeared in greater numbers on these sections as compared to the root sections with surface disinfection. Inoculation studies Table II shows the source of the isolates which were used in the greenhouse studies. The soil used in greenhouse flats to grow white pine seed and seedlings from the woodsplot was infested with the different Fusar- ium isolates. These isolates were grown on cornmeal ver- miculite medium and also on a synthetic liquid medium. The microorganisms which were reisolated from the seed- lings grown in the infested soil are shown in Tables IX- XII and Figures 10-13. The reisolation results from seed- lings grown in the first greenhouse series are shown in Table X, and the results from the second greenhouse series appear in Table XI. Plate VII shows the growth on PDA medium of Fusarium reisolated from white pine seedlings of the second greenhouse series. The reisolation results of the first and second greenhouse series were combined into one table, and they are shown in Table IX. In Table XII are listed the microorganisms reisolated from the seedlings which were grown in soil in the third green- house series. Nematode studies Tables XIII and XIV list the genera of nematodes found by nematolegists from soil and white pine seedlings grown in the nursery. Five species of nematodes con- sidered parasitic on white pine are also shown in Table XIV. The number of nematodes extracted from soil samples collected from different areas in the nursery during the 1957-1958 studies are shown in Table XV. In Table XVI is shown the number of nematodes extracted from the roots of diseased white pine seedlings collected during this study. ()1 Q I Table I. Frequency of microorganisms isolated from white pine nursery seedlings showing various degrees of root rot.8 Microorganisms Number of Percentageb isolated times isolated Fusarium 565 50.84 Penicillium 154 8.41 Trichoderma 122 6.66 Rhizopus 121 6.60 Bacteria 75 4.09 None (sterile) 54 2.95 Hormiscium 5 1.56 Alternaria 20 1.09 Z'gorhynchus 15 0.82 Rhizoctonia 9 0.49 Verticillium 5 0.27 Aspergillus 4 0.22 Chaetomium 5 0.16 Botrytis 2 0.11 Cephalotbecium 2 0.11 Curvularia l 0.05 Phoma l 0.05 Non-sporulators 654 55.70 Isolations from 1852 diseased white pine seedlings from June to September of 1957 and June to September of 1958 b Expressed as percentage of total isolations E PENICILLIUM TRICHODERMA RHI ZOPUS BACTERIA ISOLATED NONE- STERILE 'HORMI SCIUM ALTERNARIA MICROORGANI SMS OTHERS l I 1 0 IO 20 3O 40 PERCENTAGE OF TOTAL TISSUE PLANTINGS — — Figure 1. Frequency of microorganisms isolated from white pine nursery seedlings showing various degrees of root rot. 39. Table II. SOIIPCC of Fusarium isolates used in inoculation tests. Location in the nurser: Isolate Block Sectiona Bed Plot Iie nti icatim number number number number number of isolates 53 l 2 2 5 Fusarium solani 42 1 2 4 12 F. moniliforme 46 1 2 3 12 F. solani 49 1 2 4 12 E. moniliforme G2 1 2 2 6 F. oxysporum a Sewn with white pine seed b By Dr. Villiam C. Snyder, University of California in the fall of 1955 Professor of Plant Pathology, Table III. Irequenoy of microorganisms isolated ”rom 1-0 white pine nursery seedlings showing various a degrees of root rot. Nicroorganisms Number of Percentageb isolated times isolated Fusarium 78 50.00 Rhizopus 27 10.38 None (sterile) 16 6.15 Trichoderma 11 4.25 Rhizoctonia 6 2.31 Penicillium 5 1.92 Alternaria 4 1.54 Bacteria 1 0.58 Non-sporulators 112 43.07 a Isolations from 260 1-0 White pine seedlings 1». , , “ Expressed as percentage of total lSOlathnS from 1-0 white pine seedlings 41. Table IV. Frequency of microorganisms isolated from 2-0 white pine nursery seedltngs showing various degrees of root rot.a Microorganisms ‘ “umber of Percentage isolated times isolated IT‘usarium 565 55.70 Trichoderma 80 7.59 Penicillium 75 7.20 Rhizopus 51 4.71 Pasteria 50 2.77 None (sterile) 24 2.22 Alternaria 8 0.74 23,}; orhynehus 8 0 . '74 Verticillium 5 0.46 Chaetomius: 5 0.28 notrytis 2 0.18 Hhizoctonia l 0.09 Phoma l 0.09 Curvularia 1 0.09 Aspergillus 1 0.09 r5 (\3 0'1 DJ CO 0 {\D .p. Non-sporulators 'Isolations 1nrom 1085 2-0 white pine seedlings Expressed as percentage of total isolations Prov 2-0 white gine seedlings 42. rr‘r Init- le V. “reouency of Microorganisms isolated ”rom I 0,..1 white pine nursery seedlings showing various ' a degrees f root rot. Nicroorganisms Kumber of IercentaLeF isolated times isolated Fusarium 75 56.14 Rhizopus 21 10.40 Hormisoium 19 9.41 Bacteria 16 7.92 Penicillium 12 5.94 Trichoderma 6 2.97 Alternaria 5 2.48 None (sterile) l 0.49 Z‘gorhynchus 1 0.49 Non-sporulators 48 25.76 Isolations from 202 2-1 white pine seedlings Expressed as percentage of total 1 white pine seedlings S olations from 2 -1 Table VI. r“requency of microorganisms isolated from 2-2 white pine nursery seedlings showing various a degrees of root rot. Vicroorganisms Number of Percentageb isolated times isolated Penicillium 26 17.69 Bacteria 25 15.65 Trichoderma 21 14.28 Rhizopus 17 11.56 Fusarium 8 5.44 Hormiscium 6 4.08 None (sterile) 4 2.72 Alternaria 2 1.56 Cephalothecium 2 1.56 Zygorhynchus 2 1.56 EE FJ H N o :3 C f o s P“ m H O . m c) Aspergillus 1 0.68 Non-Sporulators 54 25.15 Isolations from 147 2-2 white pine seedling Expressed as percentage of total isolations from 2-2 white pine seedlings 44. sHmCdo m. fizmmdwca wdmnzodow on QwawdeSe mmo mdoow. A mzadodm HS vmdmdwxommm Hddwomdm «Odmw spaced 0% moodwwsnm wees awwo: dpmmzo vwmsdwdmm sodo ammo V 0-0 Ammov muo “Homuv . a T e O t S f o muH Amomv e no I - A m-m Aeeev - n n n 0 Ho no we ,Jso wodomddmmm ow dwmmzo vwmsdpbmm mwmsdm m. ZOSumboscheOHm wonCwSQM o: awwwedoSd mme mdoow. 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Azzadod H5 bmdoEdZommm Hsdmomdmm donmw ozadod ow moodwwzmm twmddwomm soda :mdmv mno Awommv N) I H Amomv mum “Heqv fidoa afiwofi dwmmpm 49. e 1% 1? wodoobdmmm om «Hmmco cwmddwsmm fiwmzdm q. wondmdwm wdeZmzou oz dwofiododw mum mdoox. Adzsdedm w: endoweremom Hsawomdm dedmw Scadod ow mmmawwdmm odes s:wo: demCm THmUdemm swam Smdov e-o Aweov m-o “Hoemv k C O t S r 1|» 0 mug Amomv m mum “Heev Ho modemsdmmo ow demSm @HmSnwsmm e M iv new Table VII. Frequency of microorganisms isolated from white . a pine woodsplot seedlings. ‘4 _. _.____ A Microorganisms Number of Percentageb isolated times isolated Penicillium 15 19.74 Fusarium 12 15.79 None (sterile) 9 11.84 Rhizopus 5 5.05 Bacteria 5 5.95 Trichoderma 2 2.65 Aspergillus 2 2.65 Alternaria 1 1.51 Non-sporulators 29 58.16 a Isolations from 76 seedlings b Expressed as percentage of total isolations prom white pine woodsplot seedlings 51 nHmCdo w. noonso53% ow SwedOOdmmZHmSm Hmowmdoa ado; axedo owns sOOthHOd mommwwomw. woSHoHHHHCE d m a ncmmnwzs 1 o S, .l m Zoom Amdodwwov S .l n a we r oafionm o o r c .l M gownmbOdzwmedm P p P L 1| q 41 J o Ho w mo 0 wodoosdmmo on demCm opennwsmm 52. Table VIII. Prequency of microorganisms isolated from white pine woodsplot seedlings transplanted O .. a into the nursery transplant beds. 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