l‘llll ! ! l I III” | i .THS ST‘SC‘ZES {3N DAMWNG-OFF OF RED HHS Thesis for fho Degree of M. S. MECHGGAN STATE COLLEGE Robert Wiiiiam Brandt 3955 h} i - .- This is to certify that the thesis entitled "Studies 0. Damping-off of Red Pine" presented by Robert William Brandt has been accepted towards fulfillment of the requirements for ”.5. degree in 'Bgt. 8' 1’1. I”? tho Major professor 25. 1’55 Eu {‘1 Date L' 0-169 STUDIES ON DAMPING-OFF 0? RED PINE By ROBERT WILLIAM BRANDT An Abstract Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfilhent of the requirement: for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1955 Approved THESIS An Abstract This investigation or’dampingborf’was mainly concerned with the penetration and subsequent breakdown of pine tissues by various causal agents. Damped-off seedlings were collected from inoculated soils containing pure cultures or gythium, Fusarium, Rhisoctonia, and a mixture of these three organisms. Stem lesions were serially sectioned and prepared slides observed for fungal development and possible anatomical barriers produced by the host. The growing of suitable plant material for histolog- ical sectioning presented an Opportunity to study fungicidal control of damping-off by the organisms involved. Fbur champ icals; Nanzate, Tersan, Fermate, and Orthocide-406 were used as dusts. 0rthocide-406 gave the best control on all the pathogens, and on a mixture of these pathogens. Tersan and Mansate gave fair control on all the infestations, but seedling survival was far below that produced with the use of the Orthocide. Fusarium caused the worst pro-emergence and post-emer- gence damping-off under all treatments. The mixture of the three organisms gave no noticeable increase or decrease in the total damping-off over individual infestations. It was shown that individuals or the three genera in- volved break down the host tissues in different'vays. £1thium~infected stems had weakly stained tissues in.the area of the hyphae, suggesting a chemical change produced by the fungal invasion. Although showing extensive deve10p- ment within the host, ghizoctonia infection did not seem to change the over-all structural appearance of diseased stems. Penetration by Rhizoctonia consistently involved the produc- tion of micro-sclerotia. The most destructive of the patho- gens was Fusarium. ‘Ihis organism produced large expanses of laterally compressed, heavily stained tissues. More recently infected stems showed large areas dissolved out by some chem- ical extrusion produced by this fungus. Complexes of organisms were rarely observed, and then only the two organisms, Ehizoctonia and Fusarium were found together in a single stem. A mixture seemed to be no more damaging to the structure of the seedlings than were the individual infestations. STUDIES ON DAMPING—OFF OF RED PINE BY ROBERT WILLIAM BRANDT A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1955 ACKNOWLEDGEMSNPS The author is grateful to Dr. Richard L. Kiesling and to Prof. Fbrrest 0. Strong for the advice and suggestions given through-out this in- _vestigation; for the use of equipment, materials, land library facilities, and for the criticism and correction of the manuscript. Acknowledgement is also made to Prof. Fbrrest C. Strong for assistance in the photographic work. TABLE cr'cosrsrrs I. INTRODUCTION . . . . . . . . . . . . . . . . . . i II. REVIEW OF THE LITERATURE . . . . . . . . . . . . 3. III. CURRENT IN’ESPIGAPION. . . . . . . . . . . . . . 6 Materials and Methods . . . . . . . . . . . 6. Experimental Data . . . . . . . . . . . . . lO. Greenhouse studies . . . . . . . . . . lO. Histological studies . . . . . . . . . 21. Discussion and Conclusions. . . . . . . . . 30. Summary . . . . . . . . . . . . . . . . . . 33. Bibliot’tr’aphyO O O O O O O O 0 O O O O 0 O O 34. Table I. II. -——- Han—w—v . _ _..___ . LIST OF’TABLES Page Fungus isolates and results of pathogenicity tests . . . . . . . . . . . . . . 10. Name and chemical composition of the fungicides used in seed treatments. . . . . . . ll. LIST OF FIGURES Figure Page I. Graph of survival percentage by chemical treatments. . . . . . . . . . . . . . . . . . 12. II. Graph of survival percentage by different organisms . . . . . . . . . . . . . . . . . . 13. III. Graph of post-emergence damping-off by Chemical treatments 0 e e e e e e s e e e e e 150 IV. Graph of post-emergence damping-off by different organisms e e e e e e s e s e s e e 170 V. Graph of pre-emergence damping-off. . . . . . 18. Plate II. III. IV. LIST OF‘PLATES Page Control by seed treatments on gythium and Fhsarium. . . . . . . . . . . . . . . Control by seed treatments on Rhizoctonia and a mixture of the organisms. . . . . . . . . 20. HistOIOgical figures of Pythium and Phearium. . 22. 26. Histological figures of Rhizoctonia . . . . . . I. INTRODUCTION Damping-off, the killing of very young seedlings by fungi, is still one of the most serious difficulties encount- ered in raising coniferous seedlings. Hartley (11) stated that the damping-off organisms are "specialized as to type and age of tissues which they attack rather than as to host". These eXperiments were concerned with the kinds of organisms attacking a specific host, and studies of the penetration and subsequent breakdown of tissues. An attempt to correlate the age of tissues with certain organisms was made. A single host species, the red pine, ginus gesinosa_Ait., a native American species of pine important on the light sandy soils of the upper Great Lakes region, was chosen. This tree species is assuming increasing importance as a tim- ber tree in the reforestation plans of both state and federal forest agencies of the region. Mr. E.D. Clifford, Superin- tendent, Chittenden Fbrest Nursery at Wellston, Michigan, in a letter to the author, listed red pins as the most important tree for reforestation in Michigan. It is able to grow on sites which in the past were considered only jack pine sites. Again citing Mr. Clifford's letter, the planting plan of the Manistee National Fbrest (as an example) calls for approxi- mately 95% red pine, and the rest white pine and jack pine. In 1954, Chittenden Nursery quoted the price of red pine seed at fifteen dollars a pound, and in the past recorded prices as high as thirty dollars a pound when the cone crOp was poor. 1. Considering that Pinug_ge§inosa seldom shows a germination of more than sixty percent under nursery conditions and that this species is known to be very susceptible to damping-off organisms (11), the losses may become substantial. In view of the continued heavy losses, and the lack of damping-off control in the nursery, a series of experiments was designed to study possible anatomical barriers produced by coniferous seedlings in resisting infection by various damping-off organisms. Little information on the histologi- cal aspects of the problem is available. Diseased material was collected and sectioned to permit studies of the deve10p- ment of various pathogens in the host, and of subsequent breakdown of stem tissue. The same material permitted a study of the fungal complexities involved in the damping-off problem. Seed treatments were also run to determine their~ effects on the organisms under test. 23 II. REVIEW OF THE LITERATURE Numerous early EurOpean plant pathologists and foresters emphasized the hazards of damping-off and its causal organ- isms (8). In the United States, Atkinson (l) was the first to make damping-off studies at Cornell. Spaulding (5;) and Jones (17) independently published on control measures in 1908, with the former author contributing an account of the etiology of the disease and a method of control using sul- furic acid. Hartley (9) in 1910 began inoculations with both Ehizoctonia and Pythium to study their effect against conifers. A little later, Gifford in Vermont (2), reported his studies in which he stated that Fhsarium alone was the cause of damping-off. Shortly thereafter Hartley concluded his preliminary inoculations of conifers and found species of Pythium, Rhizoctonia, and Ensarium to be the only consistent pathogens (13). Spaulding (25) working with forty-four strains and species of Fusarium in pure culture, found them all capable of causing damping-off and well over half of these were very aggressive parasites. Graves (6) inoculated healthy seedlings with Cylindrocladium scgparium, but obtain- ed no signs of pathogenicity. Hartley and Bruner's studies p (12), on the causal agents and their relationships showed that Rhizoctonia was capable of attacking seedlings too ma— dture to be killed by either Elthium debaryanum or Fnsarium moniliforme. 3. Hartley (ll) wrote an excellent review of damping-off. He discussed the economics of the problem, the value of disease as a selective agent versus the unpredictable losses, which in turn cause a lack of production stability. After this evaluation, many articles on control measures followed ever a period of a few years. Most of these ($,l4,ld,1é,é§, ég,gé) were published records of damping-off counts in vari- ous sections of the country, and the results of using vari- ously applied chemical compounds. Rathbun-Gravatt (18) demonstrated the ability of numer- ous strains of Eythium, Rhizoctonia, and Ensarium to reduce sprouting of coniferous seed. She concluded that the fungi reducing sprouting were the ones that caused decay of radi- cles. Eliason (4), testing the pathogenicity of Pythium, found that of twenty-two species and strains, all but three were able to cause damping-off in seedlings. Roth and Biker (2l,2;,2g) succeeded in distinguishing between the field symptoms of damping-off exhibited by Pythium and Rhizoctonia, and made careful studies on the temperature and moisture re- lations of the two causative agents as they affected red pine. Their main conclusions were that factors favoring one fungus discouraged the other. Specifically Rhizoctonia, was encour- aged by dry soil and warm weather, while Pythium was favored by more moist soil and cool weather. Ihey excluded Ensarium from their studies as preliminary tests showed it to be neg- ligible in the Plainfield soils with which they were working. 4. Tint (26) working with Fhsarium found that he obtained great- er pre-emergence losses with this organism than either Pyth- $22.0” Rhizoctonia, and the post-emergence damping-off was highly comparable to these latter fungi. In temperature studies with FUsarium damping-off of red pine, the fungus damage was found to be directly related to temperature. Cox (3) investigated a disease situation in a Delaware State Fbrest nursery and found a complex of Cylindrocladium scepar- ium, Rhizoctonia solani and a species of Pythium causing se- vere losses in red and white pine beds. III. CURRENT INVESTIGATIONS Materials and Methods One year old red pine seed was obtained from the Dunbar Fbrest Nursery of Michigan State College at Sault Saint Marie, Michigan. Dissected seeds proved one hundred percent viable and further tests demonstrated ninty-eight percent germina- tion in sterilized soil. Damped-off seedlings from Professor F.C. Strong's fun- gicidal test plots were collected and isolates of the damp- ing-off organisms were obtained. The diseased stems were washed in tap water and then surface sterilized by placing them in a commercial sodium hypochlorate solution for three and one-half minutes. The stems were placed on potato dex- trose agar, and incubated at room temperature. Pythiaceous cultures were purified by subsequent transfers to sterile water plates containing hemp seeds. Transfers were then made from the outer edges of all young, actively growing colonies to new plates which were incubated and stored under refrigeration. To test the pathogenicity of the various isolates, three inch clay pots of nursery soil were sterilized for one and one-half hours on two successive days. All soil used through- out this investigation was collected from the Bogus Forest Nursery at Michigan State College. At the end of the second sterilization period, the sterile pots of soil were placed immediately in sterile petri-halves filled with sterile wa- ter. After the pots of soil were cool, an agar plug contain- ing the fungus under test was placed in the approximate cen- ter of the potted soil. This was covered slightly with ster- ile soil, and fifteen red pine seeds were placed on this sur- face, and covered with one-quarter inch of sterile soil. The moisture content was kept up, to permit growth of the fungi, by frequent additions of sterile distilled water applied from below. Uninoculated checks were planted and treated the same as inoculated pots. After pathogenicity tests were completed, the non-pathogenic cultures were discarded. A survey of the pathogenic cultures indicated that EXEEf ium debaryanum, ghizoctonia solani_(Corticium vagpm), and species of Fusarium were commonly isolated and most frequent- ly proved pathogenic. Further experiments were limited to these three fungi. Inoculum was maintained on media consist-_ ing of corn meal and vermiculite instead of sand (20). The base material was steamed for one hour, 250 cc. aliquots were placed in liter erlenmeyer flasks, and autoclaved for one and one-half hours. After cooling, three one-half inch squares from a single pathogenic isolate were aseptically introduced into the medium. The prepared flasks were incu- bated at room temperature for a period of approximately five days. The ghizoctonia, Bythium, and Fusarium inocula were mixed with thoroughly steamed soil at the rate of 100 cc. to 15,000 cc. of soil. Fbr mixed cultures about thirty-three cc. 0f each of the three fungal inocula, were mixed together in a flask and then mixed as above in a flat of soil. Chem- ically treated and untreated red pine seeds were planted at a depth of one-quarter inch in the various inoculated soils as well as in uninoculated checks and naturally infested field soil. As emergence began, careful notes were taken on the rate of emergence, density of stand, and occurrence of damping-off. Damping-off checks were made at daily intervals, and diseased material was collected for histological studies. Seedlings which were damped-off, and those which wererchem- ically injured were differentiated according to the discrip- tions of Hartley (11), and Roth and Riker (21). A few healthy stems were collected to serve as checks. Counts were continued for a month, after such time the seedlings were resistant to the damping-off disease. The stems that were collected were immediately and care- fully washed in flowing tap water, and killed and fixed in FAA (15). After a minimum of forty-eight hours in the above solution, dioxane (1,4-dioxan) was used at successive one- third, two-thirds, and full strength as the dehydration med- ium. The dioxane was replaced with absolute tertiary butyl alcohol in a series of changes over a two-day period, the last change using tertiary butyl containing eosin stain. In— filtration was started by using a 56° to 58° melting point 8. parowax in a volume equal to that of the last tertiary butyl solution, and placing the Open vials in the embedding oven for twelve hours. The solution was replaced with two com- plete changes of fresh parowax at three hour intervals, with the last such change being replaced by an equal melting point tissue mat. This solution was left for two hours after which the blocks were cast. The embedded stems were then mounted on wooden blocks and serial sections were cut at twelve microns. The sections were mounted on three inch glass microscOpe slides with Haupt's adhesive (15) and numbered consecutively for each stem. I A modification of Conant's quadruple stain (15) was used for all staining. This stain contains thionin or Lauth's violet (0.1. No. 920) in place of the crystal violet in the Conant stain (2). The cover slips were cemented to the slides with HSR synthetic resin. 9. EXperimental Data Greenhouse studies. The fungal isolates showed differences in pathogenicity. Table I lists the isolates and results of the pathogenicity tests conducted with these isolates against Table I Fungus isolates and pathogenicity FUngus Identity of Fangms Soil treatment at Pathogen- line time of isolation icity 100 Rhizoctonia solani TMTD + 101 ” ' Crag-658 + 102 ' " Manzate + 103 " " Manzate + 104 " " Manzate + 105 " " 0rtho-75 + 105 ' a Crag-658 o 107 Fusarium species Sulfur 150 + 108 " ' 5400. - 109 ' ' Crag-658 + 110 Pythium sp. Ortho-75 + 111 Pythium debaryanum Ceresan M + 112 Unidentified ascomycete Check - 113 Pythium sp. 5400 - 114 Pythium debaryanum 0rtho-406 + 115 Cephalosporium sp. Crag-658 4 116 Fhsarium sp. TMTD - 117 Fusarium sp. Tersan 4 118 Curvularia sp. TMTD - 119 Penicillium notatum Manzate - 120 Mucor sp. Check - 121 Rhizoctonia solani Sulfur 150 + 122 Nigrospora sp. Ceresan M - 123 Unidentified fungus Sulfur 300 - 124 Penicillium sp. 5400 - 125 Phearium sp. Tersan ’ 126 Pythium sp. Tersan - red pine. Only Rhizoctonia, ghsarium, and Pythium were path- OSenic. 0f the twenty-seven isolates nineteen were within 10. these three generic groupings. All eight Rhizoctonia iso- lates, three Fhsarium isolates,and four isolates of Pythium proved highly pathogenic on red pine. These fifteen isolates proved to be pathogenic when soil infested with cultured inocula was planted with red pine seed. The growing of seedling materials for histological sec- tioning, provided an excellant Opportunity to test a few seed treatment fungicides in soils infested with known path- ogenic organisms. The fungicides used, their chemical compo- sitions and their manufacturer are given in Table II. All Table II Name and chemical composition of fungicides used in treatment of seeds of Pinus fiesinosa Name Chemical Composition Manufacturer Tersan Tetramethylthiuram disulfide E.I. duPont Co. (50%) Fbrmate Ebrricdimethyldithiocarbamate E.I. duPont Co. Manzate Manganeseethylenebisdithio- E.I. duPont Co. ' carbamate Orthocide-406 N- trichloromethylthiotetra- California Spray hydrOphthalimide (50%) Chemical Co. compounds were used as dust, and excess dust was removed from the seed lots by screening. The results of these chemical tests are given in the following paragraphs. All results were computed at the end of four weeks following the first 11. "" Figure I Comparison of survival of red pine six weeks after p1anting¢date. Similar seed dust treatments were used against each soil infestation. Manzate Tersan Eythigm' Control Fermate 0rthocide Manzate Tersan Fhsarium Control Fbrmate 0rthocide Manzate Tersan ' fihizoctonia Control Fermate 0rthocide Manzate Tersan @3333 Control Fbrmate 0rthocide Manzate Tersan Field_§pi;_ Control Ebrmate ‘ 0rthocide 0 20 40 6O 80 100 —_ Survival percentage by different organisms 12. ‘ ._.._.......__.._.- _"-I" Figure II Comparison of survival of red pine six weeks after planting date. Variation of survival in different soil infestations plotted for each seed dust treatment. ‘__-— v v——v— ‘ Pythium FUserium Manzatg Rhizoctonia -——-— Mix t ure2 Field soil Pythium Fusarium Tersan Rhizoctonia Mixture Field soil Pythium Ensarium Rhizoctonia Mixture ., Field soil Pythium Fusarium Ebrmate Rhizoctonia Mixture _ Field soil Pythium Fusarium Rhizoctondm Mixture Field soil Control 0rthocide O 20 $0 60 80 Survival percentage by chemical treatments. 13. seed emergence and six weeks after the planting date. In a comparison of seedling survival, the percentages of stand at the termination of the greenhouse studies were plotted for the different fungicides and soil treatments (Figures I and II). After six weeks, seeds treated with 0rthocide - 406 showed the best survival rate averaging seventy percent against all five soil treatments. Fermate gave poorest re- sults of the chemicals used but the untreated seeds suffered the worst damping-off, with an average survival of ten per- cent against the different organisms. All of the seed treat- ments afforded gpod damping—off protection in the field soil. Of the untreated seed in the field soil only one percent of the plants remained at the termination of the experiment. FUsarium infested soil yielded the least survivors. Only 0rthocide gave moderate results with the other seed treat- ments never carrying over seventeen percent of the planted seed past the first six weeks. The untreated checks sown in flats containing the dif- ferent fungal infestations, failed to show a survival of six percent except in Pythium treated soils, which allowed a thirty-nine percent survival (Figure II). Survival of Manzate and Tersan treated seeds was spotty, both chemicals being fairly ineffective against Ensarium and the mixture of organ- isms. Fermate was also very ineffective and gave fair re- sults only in the field soil where the potential inoculum 14. Figure III Post—emergence damping-off in different soil infestations. Plotted for each seed dust _‘ trgggment. ~— w w. —- -‘.-"- Pythium FUsarium Manzate Rhizoctonia Mixture Field soil Pythium Fusarium Tersan Rhizoctonia Mixture: Field soil Pythium FUIarium Contggl Rhizoctonie Mixture Field soil Pythium FUsarium Ebrmate Rhizoctonia Mixture Field soil Pythium Fusarium 93thocide Rhizoctonim. __. Mixture Field soil O 20 40 60 80 Percentage of post—emergence damping-off. l5. was very low in comparison to the manually infested flats and the soil seem to remain more moist. Figure III shows the actual post-emergence damping-off in each treatment. Since pre-emergence damping-off was also important in this eXperiment, the survival graphs fail to show the cause of mortality. Seeds treated with Manzate for example showed a seedling survival of only seventeen percent against FUsarium and yet the post-emergence damping-off as shown by Figure III only mnounted to fourteen percent, leav- ing a total of sixty-nine percent of the expected emergence unaccounted for. Pre-emergence damping-off was one of the responsible agents. Post-emergence damping-off was most severe in Fermate- treated seedlings, with Fusarium and 2132122 causing the greatest loss. Late damping-off was consistently higher in soil infested with Pythium (Figure IV). The two greatest losses in Fusarium infested soil were in seed dusted with Fbrmate and seed receiving no treatment. Total losses in the Fusarium infested soil were slightly lower than those caused by Pythium. Rhizoctonia and the mixture of infested soils produced losses up to twenty-five percent total crOp. Field soil produced a negligible amount of post-emergence damping-off. However, pre-emergence damping-off was severe in some cases. The consistent failure of any of the fungicides tested to prevent pre-emergence damping-off was noted (Table VII). 16. However, the extremely heavy losses in the untreated controls (Plates 1 and 2) shows the advisability of continuing chem- ical treatments for a partial check of losses. Pre-emergence damping-off losses were fairly uniform through-out the vari- ous soil infestations and fungicidal treatments. Only 2132? $22 failed to reduce emergence drastically, and yet produced an average damping-off of twenty percent against all fungi- cides. 0rthocide maintained a good stand of red pine through the first six weeks, but 0rthocide treated seed suffered an average pre-emergence loss of twenty-five percent against the different damping-off situations used. Figure V Comparison of the different seed dust treatments on pre- emergence damping-off of red pine planted in variously infested soils. v ‘.-*- fianzate Tersan PithlEm ‘ Control ‘ Ebrmate 0rthocide ‘ Manzate Tersan ggsarium Control Ebrmate 0rthocide Manzate Tersan Rhizoctonia Control Fermate 0rthocide Mdnzate Tersan Control Ebrmate 0rthocide ngture Manzate Tersan Control Furmate 0rthocide O 20 4o 60 80 100 Field soil Graph of pre-emergence damping-off 18. Plate I (Seed dust treatments reading downward). Pythium Control Upper left Lower left Manzate 0rthocide-406 Manzate 0rthocide-406 Check Check Fermate Tersan Fermate Tersan Ensarium Control Upper right Lower right 0rthocide-406 Tersan 0rthocide-406 Tersan Check Check Manzate Fermate Manzate Fermate _— _— Plate I 19. Plate II (Seed dust treatments reading downward). Bhizoctonia Control Upper right Tersan Tersan Check Fbrmate Fbrmate Mixture of organisms Upper left Manzate Tersan Check Fbrmate 0rthocide-405 Lower right Manzate Manzate Check 0rthocide¥406 0rthocide-406 Naturally infested soil Lower left Manzate Tersan Check 0rthocide-406 Fbrmate Plate II A ' c ' ‘: .1". . " “ ”L“? .- 76",!017. ;' r ’i‘": 7: 3+5 3??" “(We“a???“Wifi‘h‘nr“ “h e , \t Histological studies. Pythium attacks the host plant just below ground line and grows upward through the stem. Initial penetration of the host tissues was not observed and the col- lected samples were always outwardly clean, with no hyphal attachments as was seen in Rhizoctonia. One instance was recorded in which a hypha was seen lying within a stomatal orifice and over the adjacent guard cell. It could not be determined whether this hyphal strand was growing out of, or into the stem, since the stem tissue beneath was already largely filled with mycelium. Once inside the stem, the fungus was inter and intra- cellular and moved both vertically and horizontally through the host. Penetration of cell walls occurred at any angle and place the hypha made contact. There was evidence of a pressure build-up, as the host walls occassionally became indented before the tissue was broken through by a small penetration peg. The host cell wall often became quite sharply indented over this peg, but usually nearly resumed its normal position after the puncture had been accomplished. Considerable swelling occurred in the hyphal tips before a wall was punctured, and immediately after the peg had passed through the wall, the hypha regained its normal size, leav- ing a definite constriction at the wall (Plate III, Lower right), Some hyphae passed through the cell from wall to wall. Series of four and five parallel walls were penetrat- ed by a single hyphal strand with little change of direction, 21. Plate III Histological figures Lower right - Hyphal constrictions of Pythium. The hypha penetrated a series of cell walls with little change of direction. The faded nuclei in the region or activity is in direct contrast to the staining in- tensity of nuclei in fihizoctonia_infected stems. Lower left - Photomicro graph of a red pine stem moder- . tion ately infested with Fhsarium. The central DOT shows numerous hyphal strands and a heavy stain through-out. The remainder of the stem exhibits the large cavities typical of tissue breakdown by Fusariumo The dark staining protoplasm and white cross-walls of the hyphae are evident. Upper left - Hyphal strands of Fusarium and fihizoctonia in a diseased The complexities of multiple infec- rarely. stem. tion existed only , Fusarium (16ft and ght) and Rhizoctonia (cen same Stems upper ri t91") are shown in the Plate III E. 22. 4 a ll: and without apparent loss of size or vigor. If the angle of approach is even slightly acute the wall of the host is pen- etrated, but the hypha then grows intercellularly until a wall is again hit directly. Strong evidence of a host-parasite relationship within the invaded cells was shown by the disappearance of nuclei and chloroplasts in the areas of mycelial advancement, while normal check plants, and areas of infested plants that were not penetrated, showed brilliantly stained nuclei and easily observable chloroplasts. This condition was in direct con- trast to Rhizoctonia infection, where such structures were always readily seen. The fungus was much branched and rami- fied the invaded area in all directions. The deposition of lignins and other hardening materials restricted the move- ment of the fungus however, for no hyphal strand was found to penetrate sclerotized tissue. Other than the natural hardening off that comes with age, there was no observable evidence of any incompatible reactions between host and path- ogen. There was no collapse of host tissue in the attempt to seal or wall-off the penetrations. The hyphal tips were very dense and vigorous in appearance, and no dead or dying hyphae were present, which might indicate toxic substances being liberated by the deteriorating host cells. Damping—off by Fhsarium was the most damaging as far as actual structural disintegration was concerned. Phe fungus caused the formation of large cavities within the host stem, 25. or in some instances resulted in collapsing and shredding of the tissues (Plate III, upper right and lower left). The hyphae themselves appeared to be less numerous through-out the host, but the actual numbers and spread were partially occluded by the intensity of the stain in the infected areas. The staining schedule times were varied considerably in an attempt to differentiate more clearly between the host tissue and fungal growth but the Conant's Quadruple stain used through-out the investigation continued to stain both host and pathogen a deep red. The chemical exudates from Fusarium apparently changed the nature of the host tissue so that normal staining reactions could not take place. Rarely, in isolated areas and on the fringes of decay areas, the hyphal strands were a light red or even pink, allowing for a more exacting study of the fungus and it's effect on the host tissues. Phe hyphae were approximately one-third to one- quarter of the size of the Rhizoctonia hyphae (Plate III, Upper left). Although the protOplasm of the fungus stained deep red, there were a few pale and quite vacuolate hyphal strands that showed unstained cross-walls. In such cases the nuclei of the fungus stained darker and were readily observed. The deterioration of the cell walls and over-all break- down of structure of the tissue made it impossible to evalu- ate the fungal alignment in the host cells, association with vascular elements, or penetration of the relatively mature cells. The bulk of the stems collected for sectioning were 24. completely disintegrated by the time the diseased seedling was recognized as infected. The material suitable for hist- ological sectioning was limited. Only four of the one hun- dred and thirty-three slides made of ggsarium_infected seed- lings exhibited suitable detail for photomicrographing. The pine stems collected from the Rhizoctonia infested soil were often covered with the hyphae of the fungus. These hyphae were quite discernible with the naked eye when the young seedlings were first removed from the soil. The base of the above ground portion of stems of such plants was us- ually white or yellowish-white, and so consistent in this characteristic that many of the damped-off seedlings from the flat of mixed inocula, and some from the field soil were marked as Rhizoctonia. Later, the slides prepared from these collections were observed and with one exception were found to contain Rhizoctonia only. The exception was from the check row of the flat of mixed inocula and this stem showed both Fusarium and §hizoctonia (Plate III, lower right). Histologically, the hyphal strands along the stem sur- faces were seen to consist of loose and appressed stringy hyphae and small knots of closely compacted, septate hyphae having the appearance of micro-sclerotia. These sclerotial structures were very abundant in the slide preparations and apparently play a large roll in mycelial penetration of the pine host. No special organs of attachment, such as appres- soria or cementing materials were observed. Sizeable 25. a». Plate IV Histological figures Upper left - Ramification of Bhizoctonia hyphae. The Christmas tree growth effect was very common. Lower left - The intercellular spread of the fihizoctonia hyphae. The picture is oriented at right angles to the normal. The longest hyphal extensions were usually aligned with the long axis of the host. Note the in- tensity of stain in the nucleus. Upper right - Sclerotial attack in Rhizoctonia solani. A stem showing penetration at Opposite surfaces. Lower right - A second example of sclerotial build -up, this time in the interior of the infected stem. Plate 1? penetration forces must exist for mass penetrations as shown (Plate IV, Right upper and lower). The number of sclerotial bodies attacking a single stem was very variable. Some areas of the surface were densely packed with six or seven small sclerotia, while other surfaces had none, one, or several widely scattered. Occasionally a stem was invaded from both sides (Plate IV, Upper right). Some areas around pene- tration points stained densely. After the fungus was established within the host, it ap- peared to penetrate the host cell walls mechanically. There were no signs of abnormal staining reactions or dissolution of host parts that might indicate chemical changes except breakdown of chlorOphyll in the stem. A fading of the chlo- r0plasts to a pale yellow-green occurred in certain stems, which accounted for the chlorosis of the stem at ground line. The Rhizoctonia hyphae were usually the same size but variations occurred which were explained by age differences. Penetration appeared mechanical, and constrictions at the wall punctures were observed in some instances but not in others. The fungus ramified through the host with no appar- ent inhibitions. The spread was ac00mplished horizontally and vertically through the host cells with equal readiness. A Christmas-tree—like growth occurred in some stems (Plate IV, Upper left) but in others great vertical extensions of dense hyphae traveled intercellularly (Plate IV, Lower left). Ihe growth of the fungus through the stem as portrayed above occurred, as in Pythium, only through succulent tissues. Any cells showing secondary thickening in spiralar deposition or accumulation were not penetrated. Certain parenchymatous cells contained a hypha which curled inside the cell until a. corner of the cell was reached, where penetration occurred. Often the pressure of the penetration hypha would displace or indent the host cell wall, but again as in Pythium, the wall distension would decrease considerably but not entirely after penetration was affected. The fungus branched less in host tissue than in sclero- tia or cultures. Septation was not as evident, but the dis— tinguishing characteristic of fihizoctonia, the slightly bul- bous base of the new branch, closely followed by a septum, was always observed. Differential staining of fungal strands was apparently due to differences in protOplasmic densities. In Rhizoctonia infection, as well as with the other two organisms used, the lack of any antagonistic reactions on the part of the host was noted. There were no hyphal frag- ments or unstained portions of mycelia (representing dead or senescent fungal tissues) which might indicate an incompati- ble host-parasite relationship. No evidences of walling off or cell collapse to step further penetrations were observed. As eXplained in the materials and methods section, in- ocula of the three organisms, Pythium, Busarium, and Rhizoc- tonia were mixed in equal portions to yield a total inoculum eQual to that induced into the other experimental flats. 28. ll Treated seeds were planted and the damping-off results were recorded in the above greenhouse studies. Observation of the slides made from the damped-off seedlings from this flat showed that the bulk of the damping-off was caused by Rhizoc- tonia alone or Rhizoctonia and FUsarium together. In the latter case infection seemed to take place at quite widely divergent locations and the two fungi to grow toward each other through the host stem. Although they approached very closely, there was no general intermingling between the hyphal strands of the two. Destruction of the stem tissue occurred in each area of infection as it did in stems having but a single infection of a specified fungus. Pythium was not observed in a single processed stem, out of a total of sixty lesioned stems collected and sectioned from the mixed inoculum tests. 29. Discussion and Conclusions 0rthocide-406 dust gave the best control of damping-off when based on survival, seedling vigor and lack of injury to seedlings. Ehe data showed a survival of fifty-one percent in the field soil and a survival of eightly percent of seed- lings in the soil with the mixed inoculum. Post-emergence damping-off usually occurred immediately after emergence‘when seedlings were most succulent. Good plants were produced without chemical injury when 0rthocide dust was used. Ortho- cide gave the best protection of seeds and seedlings against the pathogenic organisms used both in pure cultures and mix- tures. Tersan and Manzate gave about the same amount of control against the test organisms. Both chemicals produced fair survival in Pythium and Rhizoctonia inoculated soils. 2317 ggigg_caused very severe losses both as pre-emergence and post-emergence damping-off in Tersan and Manzate treatments. The apparent percentage of damping-off in Manzate treatments may be partially due to chemical injury. Fbrmate treatments gave poor control of damping-off with a final survival of fifty-one percent against the na- turally infested field soil, but a high survival of twenty- five percent in soils infested with all of the pathogens. On seed dusted with this chemical, ggsarium caused seventy- two percent postvemergence losses, and Pythium caused sixty Percent loss, based on eXpected field emergence. 30. The untreated checks showed variations in damping-off. gusarium caused a post-emergence loss of sixty-seven percent. One reason for the large losses was the relatively heavy inoculum used in preportion to the amount of soil to be in- fested. These consistently heavy losses in all the fungicide tests, indicated that the amount of inoculum used was much greater than that normally found in infested nursery soil. However, such heavily inoculated soils resulted in severe tests which showed differences in the effectiveness of the chemicals tested. The effects of environment on these pathogens was re- ported by Tint (26), and Roth and Riker (21,22,23). Under different soil and environmental conditions, both organisms and chemical compounds might have reacted quite differently. Pythium and Rhizoctonia caused little or no structural differences in infected stems in the earlier stages of the disease. Pythium infested stems failed to show brilliantly stained nuclei in the areas of heavy penetration, the visible nuclei being pink-gray in color and highly granular. In the lesions caused by Pythium the number of chlorOplasts had de- creased, and nuclei failed to stain normally. This staining reaction, and the water soaked appearance of the lesions demonstrate physiologic changes. Stems containing Rhizoctonia showed typical red-stain- ing nuclei even when these structures were in close proximity to the hyphal strands. A chemical breakdown of the 31. chlorOplasts was apparent in the majority of the slides. In the areas of advanced hyphal penetrations the chlorOplasts were a pale yellow-green. When seen in the field the over- all effect is one of chlorosis. A characteristic of the Rhizoctonia infection was the production of micro-sclerotia. These minute knots of hyphae appear to be a common mode of establishment of the pathogen in the host, and were observed singly and in large numbers on various diseased stems. An occasional sclerotium was ob- served with the pine tissue. ‘Eggggigm_caused extreme breakdown of host tissues. Heavily stained disintegrated masses of tissue were found in slides prepared from stems showing only incipient decay upon collection. All the various tissues were attacked by the fungus. In the few places where the attacking pathogen was observed, the host was largely dissolved away, leaving the Fusarium.hyphae free in the large open cavities. The cell walls were the last structures to disintegrate, but this re- sistance might be caused by their mass rather than any cer- tain chemical resistance. A complex of organisms was observed in stems grown in soil infested with a mixture of gythium, Fhsarium, and Rhizoc- tonia. This complex, however, was not as involved as some seen earlier in sections of naturally infected Austrian pine, Prepared by the author for classroom use. The damped-off seedlings above showed unknown hyphae of two definite 32. organisms inter-twining and ramifying through-out the host cells. 1n the few cases, where more than one organism was observed in sections of diseased red pine stems, the mixture was always one of Ehizoctonia and Fusarium. They exhibited no interplay of hyphae strands. Breakdown of tissue contain- ing such a fungal mixture was accomplished by individual action of organisms and not by collective action. 35'. Summary A summarization is made, with full realization that the results are dependent on the inherent variables in such an experiment. 1. 0rthocide-406 gave the best control against all patho- gens, and against a mixture of these pathogens. 2. Fusarium caused the worst pre-emergence and post-emer- gence danping-off against all treatments. 5. A mixture of the test organisms did not increase or de- crease the total damping-off as compared to individual in- festations. 4. Individuals of the three genera used in this experiment break down plant tissue in different ways. 5. In soil infested with a mixed 1:1:1 inoculum, Rhizoc- tonia was most frequently found in diseased plants grown under environmental conditions presiding at the time the experiment was preformed. 6. Only rarely were Rhizoctonia and Fusarium observed to- gether in the same diseased stem. None of the other pos- sible combinations of organisms were observed. 7. It is highly desirable that further histological studies be made with different host species and with various other known damping-off organisms. 1. 2. 4. 8. 9. 10. 11. 12. -Abstracted in Science, Bibliography Atkinson, G. F. 1894. Damping-off. New York (Cornell) Agr. Expt. Sta. Bill. 94: 301-346, 1111130 Conn, He J. 1946. Biological Stains. Geneva, New York. Cox, R. S. 1954. C lindrocladium sco agigm_on conifer seedlings. Agr. Expt. Sta. U. of elaware Tech. Bul. 501. Eliason, E. J. 1928. Comparative virulence of certain strains of P thium in direct inoculation of conifers. Phytopath 18: 561-567. Gifford, C. M. 1911. The damping-off of coniferous seedlings. Utah Agr. Expt. Sta. Bul. 157: 145-171, 10 figs., 4 p1. Bibliography p. 171. Graves, A. H. 1915. Root rot of coniferous seedlings. PhytOpathology 5: 213-217. Hansen, T. S. 1925. A study of the damping-off disease of coniferous seedlings. Minn. Univ. Agr. Expt. Sta. Tech. Bul. 15: 1-55. Harti R. 1894. Tgxtbook of the diseases of trees. Translation by W. Somerville. Rev. and ed. by H. M. Ward. 551p., 11111. 0 London. Hartley, C. 1910. Notes on some diseases of coniferous nursery stock. n.s., v. 51, no. 799, p. 659. Hartley, C. 1915. Injury by disinfecta sandy soils. U.S.D.A. Bul. 169: nts to seeds and roots in 1-55. Hartley, C. , 1921. Damping-off in forest nurseries. 954: 1’99 0 U.S.D.A. Bul. Hartley, 0., and Bruner, S. C. 1915. Notes on ghizoctonia, PhytOpathology 5: 75-74. 54. AJ '1‘?"“" "' 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 25. 24. Hartley. C., and Merrill, T. C. 1914. Preliminary tests of disinfectants in controlling damping-off in various nurser soils. Ph t 4: 89-92. y y Opathology lgzvflartIGY. C., and Pierce, R. G. ' The control of dampin -off of conife q UOSoD.A. Bul. 453: 1-32. 8 P0118 seedliane Johansen, 1940. Plant Microtechnique. McGraw-Hill Book Co. Inc. New York and London. Johansen, L. P. V., and Linton, G. M. 1942. Experiments on chemical control of damping-off in Pinus resinosa Ait. Canad. Jour. Res. Sect. C., Bot. Sci. 20: 559-569. Jones, L. R. 1908. The dampingeoff of coniferous seedlings. Vt. Agr. Exp. Sta. Ann. Rpt. (1906-1907) 20: 542-547. Rathbun-Gravatt,.Annie E. 1951. Germination loss of coniferous seeds due to parasites. Jour. Agr. Res. 42: 71-92. Riker, A. J., Gruenhagen, R. H. and Roth, L. F. 1945. Control of damping-off of nursery pine seedlings. Abs. in PhytOpathology 55-11. Riker, A. J., and Riker, Regina, 8. 1956. Introduction to research on plant diseases. Planographed by John S. Swift 00., Inc. New York. Roth, L. F2, and Hiker, A. J. 1945 (a). Life history and distribution of P thium and Rhizoctonia in relation to danping-off of red pine seed- lings. JOur. Agr. Res. 67: 129f148, illus. ‘ Roth, L. FL, and Riker, A. J. 1945 (b). Influence of temperature, moisture, and soil reaction of the damping-off of red pine seedlings by Pythium and Rhizoctonia. J. Ag. Res., 67: 275-295. Roth, L. F3, and Riker, A. J. 1945 (c). Seasonal develOpment in the nursery of damp- d ing-off of red pine seedlings caused by P thium an Rhizoctonia. Jour. Agr. Res. 67: 417-451. Spaulding, P. 1908. The treatment of dampi lings. U.S.D.A. Bur. Plant Indus. Cir. ng-off in coniferous seed- 4: 1’4. 55. NC 25. 26. 27. 28. 29. Spaulding, P. 1914. The damping-off of coniferous seedlings. Phytopathology 4: 75-88. Tint, H. 1945. Studies in the Fhsarium damping-off of conifers. I. The comparative virulence of certain Fusaria. II. Relation of age of host, pH and some nutritional factors to the pathogenicity of Fhsarium. III. Relation of temp. and sunlight to pathogenicity of Fhsarium. PhytOpathology 55: 421-459, 440-457, 498-510. 'I'oumey, J. W., and Li, T. T. 1924. Nursery investigations with special reference to damping-off. Yale Univ., School Forestry Bul. 10, 1-56. Wiant, J. S. 1929. The Rhizoctonia damping-off of conifers, and its control by chemical treatment of the soil. N.Y. (Cornell) Agr. Expt. Sta. Mem. 124: 1-64. Young, G. Y., Davis, W. C., and Latham, D. H. 1957. Control of damping-off of conifers (abstract) PhytOpathology 27: 144. 56. am USE 0w ’I-“w‘w ‘13:" ntzi‘a‘.’ “0'42 SMILIHVV Calf i,f;"e?,.i Apr4'56 M2756 (i'iayl‘i in. n ( "‘I' '\..\1,—- 9):. .1 -. g “4 J VJ -.v(, I iun12'56~ Jul 1'9 "36 Aug 2 ‘5‘ Augie '56 Aug30 ‘56 I ‘I: I' QC"): .6 ~30 SEP 3 7' 41960 J! 1.... (101-251960 ~" ‘~ (III: II