WILT OF COTTON: STUDIES OF POSSIBLE SEED TRANSMISSION

Item Type text; Dissertation-Reproduction (electronic)

Authors Allen, Ross Marvin, 1917-

Publisher The University of Arizona.

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Link to Item http://hdl.handle.net/10150/289699 VERTICILLIUM WILT OF COTTON: STUDIES OF POSSIBLE SEED TRANSMISSION

by Ross M. Allen

A. Thesis submitted to the faculty of the Department of Pathology in partial fulfillment of the requirements for the degree of DOCTOR OP PHILOSOPHY in the Graduate College, University of Arizona

1953

Approved!. ______, 20,1933 ^ Director of Thesis QDate This thesis has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the Library to be made available to borrowers under rules of the Library. Brief quotations from this thesis are allowable without special permission, provided that accurate aclmowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the dean of the Graduate College when in their judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED:-

11 TABLE OP CONTENTS SECTION PAGE

Introduction ...... 1 Review of the I,i terature ...... 4 History . . . 4 Importance ...... 6 Distribution and Losses * ...... 6 Host Range . 10 Symptoms .. 10 On Mature ...... 10 On Seedling Plants ...... 15 Variations of Expression 15 The Pathogen 17 ...... 17 Morphology ...... 17 Physiology ...... 21 Temperature Relations 21 Influence of p H ...... 26 Nitrogen Source ...... 26 Carbohydrate Source ...... 29 Enzyme and. Alkaloid Influence ...... 30 Effect of Dyes ...... 31 Cause of Wilt ...... 31 Pathogenicity ...... 33 Correlated with Morphology...... 33 Temperature Influence 35 Cross-inoculation Experiments ...... 36 Mode of Attack ...... 40 Transmission and Spread ...... 41 By Seed ...... 41 By Root Contact ...... 44 By Vegetative Propagation ...... 45 By Plant Debris ...... I . . . 45 By Cultural Practices ...... 47 By Miscellaneous Means ...... 48 Environmental Factors Affecting the Disease . . . 48 Soil ...... 50 Soil Moisture ...... 52 Soil and Air Temperature ...... 53 Soil Reaction...... 55 Soil Fertility ...... 56 Soil Microflora ...... 58 Crop History ...... 59 Vertical Distribution of Pathogen ...... 60

iv SECTION PAGE Control of the Disease ...... 60 Resistant Varieties 60 Cultural Practices ...... 63 Soil Amendments ...... 65 Chemical Disinfection ...... 65 Seed Treatment ...... 66 Legal Measures ...... 67 Methods and Results . . 68 Collection and Description of Cultures . 68 Preliminary Experiments with SxP Seeds ...... 71 Histological Studies of Verticillium-inoculated S e e d s ...... 74 Pathogenicity Test TCsing Verticillium-infested Oats 76 Laboratory Studies ...... 79 Seed Inoculations, 1950 79 Seed Inoculations, 1951...... 85 Seed Inoculations, 1952 ...... 87, Field and Greenhouse Studies ...... 92 Greenhouse Plantings- Inoculated Seeds, 1950 . . , 92 Field Plantings- Inoculated Seeds, 1950 ..... 99 Field Plantings- Inoculated Seeds, 1951 102 Greenhouse Plantings- Inoculated Seeds, 1951 . . . 109 Field Plantings- Inoculated Seeds, 1952 ..... 112 Greenhouse Plantings- Inoculated Seeds, 1952 . . . 115 Field Inoculations, 1951 ...... 117 Greenhouse Inoculations, 1951 ...... 141 pathogenicity Test by Hypodermic Inoculation . . . 145 Culture of Young Bolls, from Verticillium- wilted Plants ...... 149 Pathogenicity Studies of Isolates Using , , and ...... 151 A Previously Undescribed Method of Isolating Verticillium ...... 152 Growth Comparisons of Isolates on Various Media . . 156 Discussion and Conclusions ...... 161 Summary ...... 174 Literature Cited ...... 177 Appendix LIST OF TABLES NUMBER PAGE 1, Additional plant hosts of Verticillium s p e c i e s ...... 11 2. Temperature relations of Verticillium species ...... •••24 3^ pH range of Verticillium spp. in culture . . 27 4* Results of pathogenicity test in artificially infested soil ...... •••••••••• 78 5. Inoculation of cotton seed by hypodermic Injection...... 80 6• Cultures of hypodermically-inoculated seeds, long-staple varieties •••••••... 82 7. Cultures of hypodermically-inoculated seeds, short-staple varieties ...... 83 8^ Drilled seed inoculated by Method B • • • • • 88 9^ Results of culturing seeds inoculated by Method C ...... 91 10. Numbers of infected cotton plants in green­ house resulting from Verticillium-inocu- lated seeds ••••••••••«••••• 94 11. Numbers of infected cotton plants in green­ house resulting from Verticillium-inocu- lated seeds ••••....••.••.. 95 12. Numbers of infected cotton plants in green­ house resulting from Verticillium-inoeu- lated seeds ..•..••....•••.. 96 13. Numbers of infected cotton plants in green­ house resulting from Verticillium-inocu- lated seeds ...... 97 14. Emergences occurring in field test, 1950 . . 101

Vi WTJMBEÎR - FAGJil 15. Hills showing emergence 14 days after plant­ ing seeds inoculated by Method B ...... 105 16. Results of cultures of Method B-inocula ted seeds on -dextrose agar ...... 108 17. Results of planting seeds inoculated by Method B in sterile soil in the greenhouse, and of culturing 21-day old seedlings . . . Ill 18. Results of field planting, 1952 ...... 116 19. Results of planting unwounded, inoculated seeds in sterile soil in greenhouse .... 118 20. Culture of branches and branch-inoculated b o l l s ...... 124 21. Culture of branches and branch-inoculated b o l l s ...... 125 22. Culture of bolls and seeds inoculated with Gliocladium throu^ boll pe d i c e l s ...... 134 23. Culture of bolls and seeds inoculated with Thomas strain of Verticillium through boll pedicels ...... 135 24. Culture of bolls and seeds inoculated with McNeal strain of Verticillium throu^ boll pedicels ...... 136 25. Culture of bolls and seeds inoculated with Tipton strain of Verticillium throu^ boll pedicels ...... 137 26. Culture of bolls and seeds inoculated with CMI strain of Verticillium through boll pedicels ...... 139 27. Location of Verticillium colonies growing from infectied seeds T ...... 142 28. Results of boll and pedicel inoculations of greenhouse plants ...... 146

vii LIST OF FIGURES (In Appendix) FUMBER 1. Photomicrograph of conidiophores and conidia of Gliocladium rossum. 2. Typical colonies of Verticillium strains and . Gliocladium on potato-dextrose agar. 3. Equipment used for inoculation of seeds by Method A, 4. Verticillium growing from wound-site and micropyle of SxP variety seeds, 5. Verticillium growing from germinated, inoculated seeds. 6. Photomicrograph of Verticillium conidiophore. 7. Photomicrograph of a portion of a longitudinal section of a Verticillium-infected seed, 8. Camera lueIda drawing of Verticillium hypha penetrating parenchyma cell-walis, 9. Ten-week old cotton plant showing typical foliar symptoms. 10, Verticillium growing from the wound-site in the cotyledon and from the fringe tissue inside the cast-off seed coat, 11, Seedlings showing Verticillium growing on coty­ ledons and cast-off seed coat.

12, Drilling apparatus used for preparation of seeds for inoculation by Method B. 13, Verticillium growing from seeds inoculated by Method C, 14, Verticillium colonies on potato-dextrose agar to which the was carried from the in­ terior of seeds by seedlihgs.

viii NUMBER

16. Growth, of Vertlcllli'um from the inoculation wound-rsite and from the chalazal end of the germinated seeds, 16. Cotyledons wounded in inoculation by Method B. Verticillium colonies at wound-sites, 17. Hypodermically-inoculated cotton stems showing vascular discoloration. IB. Modified kitchen strainer used for surface- sterilizing seeds. 19. Gliocladium cultured from pedicel and receptacle of a boll. 20. Locks of lint from bolls inoculated through the pedicel. 21. Photomicrographs of pseudosclerotia in and on lint fibers. 22. Photomicrographs of resting mycelium in and on lint fibers. 23. Gliocladium-invaded lint fiber. 24. Gliocladium on septa and placental column of cotton boll. 25. Wilted cotton plants 11 days after hypodermic inoculation. 26. Cotton plants 13 days after hypodermic inocu­ lation. 27. Growth of 4 isolates of Verticillium and 1 of Gliocladium on wort agar. 28. Growth of 4 isolates of Verticillium and 1 of Gliocladium on potato plugs. 29. Growth of 4 isolates of Verticillium and 1 of Gliocladium on Czapek’s Solution Agar.

vix ACKNOWLEDGEMENTS

Grateful acknowledgement is made to Dr. J. G. Brown who directed the thesis. The author is indebted to Dr. R. B. Streets, Dr. Alice M. Boyle, and Dr. P. D. Keener for critical reading of the manuscript and for their helpful suggestions. Greenhouse space was generously furnished by Professor W. E. Bryan, Head of the Department of Plant Breeding, Acknowledgement is made also to Dr. W. S. Phillips, Head of the Department of Botany and Range Ecology; to Professor E. H. Pressley, Professor of Plant Breeding; to Professor R. E. Seltzer, Associate Professor and Acting Head, Department of Agricultural Economics; to Smith Worley, Jr., Assistant Plant Breeder; and to Edward L. Breazeale, Assistant Agricultural dèemist, for valuable aid. Approximately one-fifth of the work was supported by a fellowship, the donor of which wishes to remain anonymous, and to whom the writer is deeply appreciative. To my wife, Louise, for encouragement and timely assistance, I express my gratitude.

iii INTRODUCTION

Verticillium wilt is now considered a major disease of cotton, especially in certain of the irrigated areas of the Southwest. Intensive culture of this crop in the Southwestern States has been spurred by the attractive profits to be gained under the present program of Federal price-support. Continuous cropping of wilt-susceptible varieties is common practice among local growers. Verticillium-infested fields are being replanted to cotton in spite of the knowledge that such practice will result in reduced yields. Substantial acreages of land never before cultivated are now being cropped with cotton of the susceptible Acala varieties. Almost three-quarters of the Arizona cotton acreage is planted to one or more of these susceptible varieties. Approximately one-half of the total irrigated land in Arizona is now devoted to cotton production. Wilt caused by Verticillium has now been reported from all of the major cotton producing regions of the State and its frequency and severity are definitely increasing. Workers in other States report a similar situation with numerous reports of new occurrences from areas in which the disease has never been found previously. 2 The manner of transmission and snbseqnent spread of the disease in an area has been studied by numerous investi­ gators, but as yet no completely satisfactory explanation has been made for the initial appearance of the disease in many regions. Indigenous occurrence in native soil has been propounded as one explanation; introduction of other hosts bearing the parasite, or of infested soil, into an area has been suggested. Seed transmission is thought possible, by some workers and is considered improbable by others. Of the methods mentioned, field observations strongly support the seed-transmission theory for several reasons; (a) the scattered occurrence of infected plants in the field, (b) new land in crop for the first time may be affected, (c) simultaneous occurrence of the disease in geographically isolated areas, and, (d) the fact that initial reports of the wilt in most cases in Arizona have been limited to cotton as the host plant. It has been considered therefore, that further investi­ gation of the possibilities of seed transmission of Verti­ cillium wilt of cotton is necessary in an effort to deter­ mine the possible role of seed in establishing initial

infections. Observance of the fungus following its inoculation into seed should provide information regarding its effect on seed germinability, the infection of seedlings grovm from inocu­ lated seed, and the ability of the fungus to remain viable 3 in dormant seeds. Inoculation of various plant parts of^

different cotton varieties with the pathogen could aid in

tracing the path of the fungus into the seed from the plant

stan as well as indicating resistance in the different

cotton varieties.

Study of the various isolates of the fungus for physiological specialization may be of value in cotton

breeding work to establish varieties resistant to the

fungus strains,especially in Arizona,and possibly be of use

in other Verticillium-affected regions as well. REVIEW OP THE LITERATURE

A review of the pertinent literature has been made in order to provide a source of information useful for this and for later work on the problem. Rudolph (144) gives an excellent summary of literature published prior to 1929,

HISTORY "The origin of cotton is shrouded in obscurity. There is no authentic history of early cotton culture,... (Duggan and Chapman, 41, p. 21), When the cotton fiber was first woven into cloth is not known, but archeological evidence indicates that cotton fibers of the arboreum type (Cos- sypium arboreum L.) were used in northwestern India to make cloth as early as 3000 B. C. Cotton fabrics have alsp been found in prehistoric pueblo ruins in Arizona (18).

Despite the long history of the economic use of cotton, it was not until 1914 that wilt of this host, caused by

Verticillium albo-atrum R. & B., was reported by Carpenter

(24*25) in two plants at Arlington, Virginia, Verticillium wilt of cotton first appeared in Arizona in 1921 but was mistakenly diagnosed by Taubenhaus as the Pusariura wilt of the South (5), True Pusarium wilt caused by P. oxysporum f . vasinfecturn has not been recorded in Arizona, Herbert and Hubbard (77) observed Verticillium hadromycosis in the 5 San Joaquin Valley of California in 1927, and Sherbakoff (15?) in Tennessee diagnosed a wilt of cotton as this disease in the same year. The Sherbakoff report mentions sporadic occurrence of the disease. Miles and Persons (118) discovered the disease in Mississippi in 1929. A survey in 193Q^1932' determined that Verticillium occurred in eight counties of that state. Cotton wilt, attributable to Verticillium albo'-atrum^ was reported as occurring in Greece in 1932 by S'arejanni

(1 5 1 ) immediately following importation of cotton seed from the United States. It had become widespread in that country by 1 9 3 8 . Occurrence of the wilt was noted in Brazil in 1933 (Viegas & Krug, 176} where appreciable economic loss in the cotton crop was being experienced. Taubenhaus (I6 3 ) observed in 1927 a cotton wilt near Waxahachie, Texas, that differed markedly from the common Pusarium wilt, and later in 1 9 3 6 , determined the cause to be Verticillium albo-atrum. He thought it significant that the new disease occurred in alkaline soils and in the same fields as those in which Texas root rot was destructive. He based this observation on the fact that Pusarium wilt had not been of considerable importance in the regions in which root rot was abundant. By 1 9 3 7 , extensive wilt of cotton had been recorded from several districts of Arizona. Brovm (.3) inspected the

Safford district and found Verticillium wilt in 11 of 22: 6 fields visited. In culturing material collected on this trip Pusarium sp. was commonly found accompanying the Verticillium in infected plants. Verticillium wilt was again evident in all cotton districts of Arizona in 1940 and was described as being very erratic in effect. The disease affected the crop little or not at all in some heavily infested fields, and in other fields of the same variety of cotton, it caused as much as 50 percent loss of yield (4-). By 194-9, Verticillium wilt had become the most important disease of cotton in Arizona (.5) • Lehman and Garriss (97) in 194-8 reported the occurrence of the disease in cotton in seven counties of North Carolina located at opposite extremes of an area extending about 230 miles. Farmers of the area thought the disease probably was present in 194-5. A severe epiphytetic of cotton wilt, causing over 50 percent infection, occurred in the province of Sul do Save, Mozambique, in 194-9, this being the first record of the disease for the area (Cabral, 23),

IMPORTANCE DISTRIBUTION That Verticillium hadromycosis is an important and serious disease may be indicated by its widespread geograph­ ical distribution. Presley (.132, p. 4-99) has stated: "Verticilliim wilt is now Imown to occur over ■ the entire Cotton Belt of the United States from the Atlantic to the Pacific. Losses from the disease, although variable from year to year in any one particular locality, appear to be constantly increasing.’*'

Miller and Nance (115), reporting in 19^9 on an incom­ plete survey, have indicated the occurrence of Verticillium in cotton land in Alabama, Arkansas, Georgia, Illinois,

Oklahoma, and Texas. On potato land, Maine and Massachu­ setts are included. The same report indicates that Verti­ cillium infestations also occur in New Jersey and New York where hosts other than cotton are affected. Occurrence in cotton in the States of Arizona, California, Mississippi,

North Carolina, Tennessee, and Virginia has been mentioned previously.

New Mexico has had serious infestations according to

Leyendecker et al. (102), where a survey of 8^ fields in the Mesilla Valley showed more than 88 percent of the fields to be wilt-infested. The mint industry in the states of Michigan and Indiana has suffered severe losses from attacks by a strain of this fungus, according to Nelson (123). Schaible et al. (.152) report Verticillium wilt is the most

important fungus disease of tomato in Utah. Potatoes are known to be susceptible to Verticillium (Nielsen, 12^;

Keyworth, 89). In addition to the United States, other countries

where Verticillium wilt affects cotton include: 8

Argentina (Fawcett, 50), China (Cheo, 29)> South Africa

(Hansford, 75)» Russia (Pan Union, 129), Peru (Barducci, 9),

Brazil (Viegas and Krug, 1?6), Greece (Sarejanni, l 5 D >

Central Asia and Bulgaria (Butler, 22), and Mozambique (Cabral, 23).

Verticillium wilt is generally worldwide in occurrence

as may be shown by listing a few of the countries from which Verticillium has been reported affecting hosts other

than cotton:: Canada (Easthara, ^2), England (Keyworth, 88),

India (Patel et al., 130), Germany (Reinke & Berthold, 136),

New Zealand (Chamberlain, 2?), Italy (Curzi, 3 D , France (Dufrenoy, 4o), Argentina (Fawcett, 4-9), Netherlands (Van der Meer, 112), and Prince Edward Island (Ayers & Hurst, 6). Losses in cotton due to this disease evidently vary

from year to year. Most severe losses are sustained in

areas continuously cropped to cotton according to Lehman and

Garriss (97)» The continuous increase of the disease has been mentioned by Presley (132). In California, Herbert and

Hubbard (.77) noted that yields of healthy plants were sub­ stantially greater than those of diseased plants, Barducci

(9), reporting from Peru in 194-2,stated that the annual loss for the country may be conservatively estimated at 2,500,000-

soles (equivalent to $1,185,000 at the 194-1 rate of exchange). Losses from Verticillium in Alabama have been quite mild, in most cases only scattered plants are affected and well 9 defined infested spots are established in some locations in which the disease may appear one year and entirely dis­ appear the next, depending largely on moisture and temper­ ature in July and August (Smith and Wilson, 15^7). Alstatt (2), in surveying El Paso and Reeves counties in West Texas, discovered that incidence of the disease in certain areas varied from 25 to 9? percent, causing reductions in yield as high as 50 to 60 percent. For El Paso county he estima­ ted an average reduction in yield of approximately 20 per­ cent and only slight reduction for Reeves county. Hie crop- damage potential is rapidly increasing. Leyendecker et al. (102) classified a group of 84 in­ fected fields: in New Mexico into five categories: those free of wilt and having no damage to the crop, those with very light infection and having an estimated damage of 0-2 percent, those moderately infested and with estimated dam­ age of 5-10 percent, and finally, those with heavy infec­ tion and having estimated damage of 10-20 percent or more. Of these 84 fields in the 1947 survey, 11.9 percent fell into the first or wilt-free category, 3^.9 percent into the second, 21.4 percent into the third, 13.1 percent into the fourth, and 9*6 percent were classed as heavily infected. In general the survey indicated less actual damage was incurred in 1947 than had been estimated in a

similar survey in 1944, but the disease was more widespread. 10 Wilt-free fields accounted for 2^.3 percent of those surveyed in 19V+ as compared to only 12 percent in 19^7 • Not only does Verticillium reduce the yield of cotton, hut it also lowers the quality of the fiber by adversely affecting the length, strength, and grade CLeyendecker, 100).

HOST RANGE In addition to cotton, Verticillium spp. have an excep­ tionally large host range. Rudolph (1^3;iM^) has prepared a host index which includes l*+0 distinct species distributed in 35 families and 18 orders. Rudolph*s host index included reports through 1928 and a few papers published in 1 9 2 9 . Since that date a number of additional hosts have been reported. These are listed in Table 1.

SYMPTOMS

ON MATURE PLANTS Carpenter (25) failed to describe specific symptoms for cotton wilted by Verticillium albo-atrum. but compares the wilts caused by this fungus and by Pusarium oxysporum f. vasinfectum in several hosts. He recognized the similarity of the two wilts and stressed the need for cultural means in determining the causal organism in each case. Miles and

Persons CII8 ), also reported that they were unable to dis­ tinguish reliable symptoms separating Verticillium wilt from

Pusarium wilt in Mississippi. However, Sherbakoff (155) TABLE 1

Additional plant hosts of Verticillium species

Scientific name Common name Author

Abroma augustum Hansford (74) Aconiturn Fischeri var, False monkshood Dimock (36) Wilsonii Acer campestre Goidanich (62) Aesculus Hippocastanum Horse chestnut Wollenweber (190) vaF. Cabbage Snyder et al. (159) capitata Brassica oleracea var. Brussels sprouts Snyder et al, (159) Remmifera Campanula isophylla Beaumont (13) ' Cannaht*F“ sat i va Hemp Vassilieff (175) Catalpa speciosa Northern catalpa (cigar tree) Carter (26) Gentaurea cyanus Bachelor’s button Dimock (36) Ceratonfa siligua Carob Miller (114) Cercis "sTliquastrum Redbud Goidanich (64) Chimonanthus fra^rana Goidanich (62) Citrus sp. Ruggieri (150)% Clematis sp. Dimock ( 36) Digitalis lanata Dimock (36) Digitalis purpurea Common"foxglove Dimock (36) Erica australis Heather Snyder et al. (159) Erica persoluta Heather Snyder et al. (159) TABLE 1 (continued)

'Scientific name Common name Author

Brytlirina caffra Coral tree Miller (114) Eupliorbia sp. - Goidanich (62) Patsia .japonica - Goidanich (62) ÿuchsla hybrida Fuchsia Baker et al. (7) Gerbera jamesonl Transvaal daisy Snyder et al. (159) Glycine Max Soybean Vassilieff (175) m .ichrysum bracteatum Strawflower Tompkins & Ark (170) Goasyplum barbadense Cotton Cheo (29) ï(oelreutâria paniculata Golden Rai n-tree Goidanich (62) Linum usitatissumum Flax (Punjab) Rudolph (145) ** Llniim usitatissumum Flax Marchai (108) # Lonicera biflora - Goidanich (62) Maolura"aurantiaca Osage orange Goidanich {63) Manihot esculenta Cassava Hansford (75) Manihot utilis'slma Cassava Hansford (73) Medicago sativa Richter et al. (139) Plea europa Snyder et al. (159) Partbenium argentatum Guayule Schneider (153) Pelargonium domesticum Geranium Baker et al. (7) Felargonlum hortorum Geranium Baker et al. (7) Persea americana Zehtmeyer (192) Pistacia vera Pistachio Snyder et al. (159) Pinus ban^ksiana Jack pine Fisher (53) *** Pinus ponderosa Ponderosa pine Fisher (53) Pinus resinosa~ Red pine Pisher (53) Phlox panlcuTata Perennial phlox Dimock (56) tremula European Aspen Liese (104; ## Raphanus sativus , icicle Snyder et al, (159) Reseda odorata Mignonette Dimock (36) TABLE 1 (continued)

Scientific name Common name Author

Rohinia {pseudo-acacia Black locust Carter (26) Salpip;log3i3 sinuata Painted-tongue pimock (36) Santolina chamaecyparissus Lavender-cotton Dimock (36) Sesamum orientale Sesame Hansford (75) Schinua terehinthefollus Brazilian pepper Miller (113) Sida spinoaa Emp. CGC (45) Sophora japonica Jap. Pagoda Tree Goidanich (64) Tephrosia sp. Hansford (75) Tilta spT Linden tree (Lime) Wollenweber (190) Triumfetta sp. Hansford (74) Viburnum lantana Wayfaring tree Baines (8) Viburnum tomentosum Baines (8) Vi^na sTnensis Baker et al. (7) Vi^a sinensis Blackeye bean Mackie (107) American Jute Vassilieff (175)

inoculations only pot tests # natural infection in Prance radicle decay of seedlings seedbed infection of seedlings

H 03 stated that the main field symptoms of Verticillium wilt distinguishing it from the wilt caused by Pusarium are the complete shedding of the leaves and noticeable shedding of young bolls before withering of the tips and branches, a distinct discoloration of the cambium, (an error, as point­ ed out by Rudolph (1^)» and frequent development of new branches at the base of the plant. Furthermore, the fibro- vascular discoloration present in both wilts seems to be more evenly distributed in the case of Verticillium infec­ tion. According to Taubenhaus Cl65) the fibrovascular dis­ coloration in the case of Verticillium wilt is confined mostly to the interior of the cylinder of both roots and stems, the discoloration being most pronounced in the lower part of the stem and progressively less evident higher on the stem, while with Pusarium wilt the discoloration is mostly in the outer woody tissue. Presley (133) specifies tliat vascular discoloration caused by Verticillium is lighter b rom than that produced by Pusarium. The first externally observable symptom of Verticillium wilt is a distinct mottling of the leaves with pale yellow­ ish, irregular areas appearing at the margin and between the principal veins. This symptom occurs in early summer, usually on the lower leaves, spreading to the middle and upper leaves of the plant later in the season. The yellow areas in the leaves gradually become paler and more whitish and necrotic, finally dying and turning brown. 1 5 At tile time or even before tiie yellow discoloration appears in the leaves, a longitudinal cut into the wood at the base of the main stalk may reveal a slight brown­ ing of the vascular system. This later becomes very pro­ nounced. In the late summer, when some plants usually die, the effect of the disease is most apparent. The leaves of badly diseased plants present a mosaic pattern of rust- colored dead areas with yellowish margins lying between narrow strips of green bordering the principal veins. Eater the leaves fall and the plant may become defoliated except for a few small leaves at the top of the plant and at the ends of branches. Resistant varieties may become infected yet show no, or only mild, external symptoms (Herbert and Hubbard, 77)•

OH SEEDLIMG PLANTS In young plants v/ith 3 to 5 true leaves there is, con­ siderable stunting according to Presley (133); leaves be­ come darker green than those of a normal plant, and appear somewhat crinkled between the veins. In the seedling stage cotyledons become yellowish, followed by rapid desiccation. Vascular discoloration is evident, particularly at the base of the hypocotyl, and infected seedlings usually die.

VARIATIONS OF EXPRESSION Differences in symptoms are reported by Miles (11?), who stated that a California strain of Verticillium caused 16 no mottling or blotching of the leaves although these symp­ toms were pronounced on cotton plants infected with a Mississippi strain.

Complete absence of symptoms in other infected host plants have been recorded by Wilhelm (180), while Torgeson

(1 7 1 )j Rudolph (1^5)s and Snyder et al. (1^9) report wilt symptoms without vascular discoloration. Richardson (1 3 8 ), working with wilt of eggplant, stated that vascular discol­ oration need not always show in all parts of a diseased plant.

In contrast, Roberts Cl^l) found that tomato plants becoming infected late in the year may show no external symptoms.

McKay (110) made substantially the same statement regarding late-infected potato plants. Keyworth (86) records two types of wilt outbreak in hops based upon -vrLde variation in symp­ tom expression and severity of disease. Infected plants suffering from nitrogen deficiency may not show typical symp­ toms of infection (Donandt, 3 8 ). Keyvrorth and Hewitt (90) had noted the same condition in hop plants and stated that, in all probability, conditions in the plant had inhibited both the growth of the pathogen and its ability to cause wilting, .in Arizona, as Bro^m (20) points out, some plants die, some are only stunted, and other plants apparently re­ cover from attack by this fungus. 17

THE PATHOGEN TAXONOMY A description of the fungus is given by Carpenter (25, p. 537) as follows: "Conidia ellipsoidal, unicellular, 4.0 to 11.0 by 1.7 to 4.2yi, abscissed singly from the sterigma tips of verticillate conidiophores. Primary whorls or vir- tels of branches, 1 to 8 in number, 30 to 9C^ apart, some­ times bearing secondary virtels. Branches 1 to 7, usually 3 to 5 in number, 13 to 38^ long, tapering, straight to slightly bowed. Conidiophores 100 to 30G^ or more in length. The terminal branch of the conidiophore is from 15 to 60yj long. Conidia may or may not collect in heads on the sterigma tips. Mycelium septate, hyaline to brown with age; may become swollen into chiamydospore-like chains of closely septate, knotted masses. These aggregates constitute the sclerotia of the fungus.” Carpenter’s description was selected because it was the first description of the fungus known to be pathogenic to cotton. The taxonomic history of the pathogen is fully dis­ cussed by Rudolph (144); Nees von Esenbeck established the genus VerticilHum in 1816. The genus Acrostalagmus. created by Corda in 1838,has been shown to be a nomen nudum by the work of Hoffman and Carpenter and the two genera are now regarded as synonymous.

MORPHOLOGY In 1879, Reinke and BerthoId (136) reported the 18 discovery of a fungus causing wilt of potato plants. They named the organism Verticillium albo-atrum Reinke & Berthold. iCLebahn C92)> in 1913, isolated from wilted plants a very similar organism which he felt was sufficiently dis­ tinct from Verticillium albo-atrum R. & B. to warrant the creation of a new species, KLebahn. The difference between the two organisms is morphological: Verticillium albo-atrum R. & B, characteristically forming only dark brown resting mycelium and never microsclerotia or pseudosclerotia, while V. dahliae is purported to form pseudosclerotia in great abundance. The controversy which has developed is, as yet, unsettled, with many workers in­ cluding Van der Meer (112), Isaac (.80), Berkeley et al. (12), Drummond (39), and Van Beyma (1?), maintaining that the dif­ ference between the two isolates is sufficient for consid­ eration as distinct species., while many workers claim the difference is insignificant and regard the isolates as strains or variations of the one species Verticillium albo- atrum R. & B. Among those defending the latter viev/point; are Bewley (1^>, Rudolph (1^^), Wilhelm (179)? Presley (133)? Goidanich (6 3 ), Kelson (121), and McKeen (111). Keyworth (8 9 ) points out that of 23 isolates made in Connec­

ticut 18 were of the microsclerotial type (V. dahliae) and k- were of the dark mycelium type (V. albo-atrum) but that 5 intermediate strains formed both dark mycelium and micro- sclerotia and thus could not be differentiated on their 19 cultural characteristics. The intermediate forms therefore lend support to the theory that dark mycelial and micro- sclerotial forms are members of Verticillium albo-atrum

R. & B. and that Verticillium dahliae Klebahn may not be a valid species. Hansen (72) attributes the variations to a heterocaryotic condition existing in the conidia though this condition has not been definitely demonstrated. Both

Hansen (72) and Presley (133), working with monosporidial cultures, have demonstrated variability in subsequent trans­ fers of the organism on artificial media.

Presley Cl33) considers that classification based upon microsclerotial formation is faulty and states that Bew­ ley *s (l^+> SIX strains of V. albo-atrum. as based on their microsclerotial production, have their counterpart in cul­ tures he has selected from sectors of a monoconidial cul­ ture and that the three forms of van Beyma Thoe Kingma '{15), based upon microsclerotial characters, also appear to be represented in the cultures derived from a single conidium.

Isaac (80), also working with monosporidial cultures, main­ tains that his microsclerotial forms have never given rise to the dark mycelial forms and vice versa, but have remain­ ed constant throughout his investigation. He describes the formation of mycelial knots resembling microsclerotia

(by dark torulose hyphae).

For clarification as to the exact morphological 20 structures.involved in this controversy Berkeley et al, (12, p, 742) have offered the following descriptions:

"Resting mycelium: Masses of dark, thick-walled hyphae with numerous crosswalls, the individual cells of which are torulose, somewhat resembling chlamydospores, or may be otherwise little dif­ ferentiated from the rest of the mycelium." "Pseudosclerotia: Mono-hyphal, thick walled, dark coloured, tissue-like formations, the result of a budding process." The term "pseudosclerotia" is suggested as the correct substitute for the names "sclerotia" and "microsclerotia", since those bodies do not consist of a weft of intertwining hyphae and have no cortical tissue differentiated from the rest of the structure. Nelson (123) considers the presence or absence of microsclerotia in cultures of V. albo-atrum

(or V* dahliae) taxonomically unimportant and of only minor value in classification. Ludbrook (106) proposed as ex­ planation for an apparent geographical location for the two forms in question: that V. albo-atrum is found in lo­ calities having cool summers but that V. dahliae is found in regions of both cool and hot summers. Morphological changes on artificial media have been reported by nymerous authors ; Rudolph (144 ), Chaudhuri

(28), Presley (131;133), Wilhelm (179), and others. Berkeley et al. (12) reported the use of potato plugs upon which the fungus was cultured to cause the fungus to regain •resting mycelium* which was lost in repeated transfers on 21 agar media. In contrast, Tompkins and Ark (I7 0 ), found that conidial and mycelial forms isolated by monosporic culture, were maintained unchanged through successive trans­ fers for many months and appear to be highly stable. Pres­ ley (1 3 1 ,1 3 3 )) noted that patch variants are produced most frequently by the microsclerotial type of culture while the appressed mycelial type is relatively constant and produces only an occasional, fluffy mycelial variant. He adds that the species V* albo-atrum. is composed of not one but many biotypes which differ greatly from one another in cul­ tural characters when grown on nutrient media, and that re­ peated selection to a type in laboratory cultures appears to reduce the number of variant types. Isaac (8 0 ) noted that all hyaline variants appeared identical in culture, and that when microsclerotia-forming capacity was once lost, it was never regained. Morphological descriptions of this fungus are numer­ ous and extremely varied. As early as 1931, Berkeley et al. (1 2 ) regarded spore size and shape, as well as mycelium and sporophore measurements, as being **^not particularly characteristic for either group".

PHYSIOLOGY Temperature Relations? Physiological reactions are as . , . - ( varied as morphological characteristics and appear to have direct influence on the morphology of isolates, Wilhelm 22 (1 8 2 ) observed that low temperatures favor the production of microsclerotia by Verticillium in dead tissues of toma­

to plants in the field. In another report, Wilhelm (179) states that a temperature difference of 3 to 6 degrees: C, within the growth range (10 to 31 degrees C.) of V. albo- atrum (only the wild type or conidial constituent, not white mycelial variants) may induce in this fungus marked divergences in cultural appearance and morphological char­ acters, especially of the resting stages. Colonies grown at low temperatures (10 to 22 degrees,C.) were jet-black and consisted almost entirely of thick microsclerotial crusts. At higher temperatures (25 to 31 degrees C.) the colonies were creamy-white and microsclerotial development was sparse. Thus he concluded that the resting structures of V. albo-atrum, particularly the microsclerotia, and col­ ony appearance were not reliable characters for specific separation. However, Isaac (80) tested the cultural morph­ ology of several isolates over an even greater temperature range (4-. 5 to 30 degrees C.) and found that wherever posi­ tive growth was recorded the morphology of the resulting mycelium was normal or true to the type of isolate employed. It is apparent from the number of reports discussing the temperature relations of Verticillium in laboratory and field work, that this factor is of prime importance in the physiology of the pathogen and probably exerts great

influence on the pathogen-host relationships relative to 23 patliogenicity, and disease occurrence, severity, and spread, Alth-ongh Rudolph (1^^) states that Verticillium hadromycosis is primarily a disease of cool regions in contradistinction to hadromycosis, the opposite condition exists for the South Central and Southwestern parts of the United States. Furthermore, the fact that cotton, which requires warm temperatures for growth, is severely affected in a number of warm, cotton-producing countries, outmodes this conclusion. Rudolph (lV+); himself points out notable ex­ ceptions occurring in the State of California. It is evi­ dent, however, that the temperature factor is limiting under certain conditions. Rudolph (144) summarizes the findings of a number of workers who have determined the cardinal temperatures for the pathogen in plants and in culture. The optimum temperature for disease development in plants (approximately 23 degrees C.) closely approximates that of the optimum temperature for growth of the parasite on artificial media. The minima listed in Rudolph's table (p. 2 3 1 ) are:: for disease to occur in plants, 1^. 6 degrees C., for growth in culture, 4-,4- degrees C. ; the maxima are 25 degrees C. and 37 degrees 0., respectively. Since Rudolph’s publication other investigators have published their results* Table 2, fashioned after that of Rudolph, indicates the variety of some of the reported observations. TABLE 2

Temperature relations of Verticillium spp.

In culture

De gre es C ent i gr ade Author Species Min, Opt. Max.

Is-aac (80) V. species .... . 4.5 22.5 fa Tmicrosclerotial type) - w 20-,32.5 (dark mycélium type) - 25 (chlamydospore type) «W 30 («) Ludbrook (106) V; dahliae.. - 30 (+) Do V. àlbô-at’i^hi - ## 28-30 Wilhelm (179) V, albo-atrum 10 ' ' -, 31 Williams (188) al'boratrum 25 30 (+) D o .... V, dahliae 25-50 30 (+) Mujic'a (120) V. albo-atrum 3r6 18-25 27-33 Verona and Ceccarelll (178) V. species 24-26 Richardson (138) V. dahliae 8 21-24 34 Patel (130) V. dahliae 12 22:5 30 Lèyehdècker (100) V. albo-atrum 5 25.5 30 Barducci and Rada (11) V, albo-atrum - 22 McKeen (111) 7. albo-atrum 8 (—) 22—24 28-32 TABLE 2 (oontinùed) In plants

Degrees Centigrade Author Speciesi Min. Opt, Max,

Ludbrook (106) 7, albo-atrum m 28 Do V. dahliae "mm ' mm 3:0-32 Richafdson (138) V: dahliae . 11 m . 30-35 Osniuh ( 10.6) 7. albo-atrum 25.5; Nielsen (124) V. albo-atrum 12.1-15.5 21-24 28.9-30 McKeen (111) 7. albo-atrum 12 20-28 32

CO OI 26 Van Koot and Wiertz (173) have shown that the thermal death point for isolates of V. dahliae from and tomato occurred at 60 degrees C . and 50 degrees C., res­ pectively, Verticillium albo-atrum, according to Sarejanni (151) has vi thstood temperatures exceeding 40 degrees C. and below 0 degrees C. and remained viable without subculturing for three years. Influence of pH; Different hydrogen-ion concentrations of artificial media produce varying effects which further confuse the observer of Verticillium reactions, Isaac (80) found a directly proportionate effect upon the forma­ tion of microsclerotia. Using an old culture of a micro- sclerotial-type isolate, he found that no microsclerotia were formed on media having a pH of 3,6, whereas at 8,0, 8.6, and 9,6, dense masses of these resting bodies appeared. The age of the colony exhibited its influence on this experi­ ment when it was noted that young cultures of the same type produced microsclerotia abundantly on each plate where growth occurred, that is, at a minimum of approximately 4,9 and at higher pH's* Verticillium spp, evidently function over a wide range of pH as indicated by the previous citation. A summary of findings by certain investigators is given in Table 3, Nitrogen Source : Isaac (80) has shown that the fungus changed the pH of media before growth started. Colonies grown on normal Dox's and potato-extract media rapidly TABLE.3

pH Range of Verticillium spp, in culture

pH Author Species:. Min, Opt, Max#

Patel (130) V. dahliae.. 2,5 4,6-5.2 9.0 Van dèr Veen (172) V, alSo-atrum 5,0-8.0 Richards bn (138) V. dahliae.. g.3 above 5,4 9,0 Verona (178) V, alhd-atrum 8.5 Do dahliae 4,9 Do V, trachelphilum 5,6 Do V, amaranti 5,0 m Isaac (80) .... V# species or strains 3,0-3,6 ™ - Barducci and Rada (11) V. albo-atrum 7.0 Haensler (71) V. albo-atrum 4,0 6.0^8.0 28 produced a high degree: of alkalinity, while on a modified Dox*s agar containing 0.2 percent ammonium nitrate the de­ gree of acidity rapidly increased. Van der Veen (172) had

shown earlier that the best sources of nitrogen for the fungus were ammonium ions or asparagine, and that cultures with a strongly acid ammonium salt rapidly reached the maxi­ mum degree of acidity at which growth was possible, while cultures containing nitrate attain an equilibrium at about pH:8.0. According to the latter report, nitrate can serve only as a source of nitrogen for the fungus when no ammonium ions are in solution. Further effects have been recorded by Isaac (80) rela­ tive to nitrogen source in artificial media. By substituting

asparagine for the sodium nitrate of Dox*s medium, it was found that M (microsclerotial). and C (chlamydospore) iso­ lates grew true to type on all test variations (0.1 percent, 0.5 percent, 1.0 percent asparagine). However D (dark mycel­ ium): type produced no resting mycelium on 1.0 percent, little

on 0.1 percent, but on 0.5 percent asparagine medium produced abundant resting mycelium. In a similar test, substituting peptone, M and C forms were true to type at all percentages; D type produced no resting mycelium on 0.1 percent, very

little on 0.5 percent, but abundantly on 1.0 percent peptone medium. All forms grew true to type on normal Dox’s medium

containing sodium nitrate with M and D types shovrLng the 29 greatest tendency to form hyaline variants on this substrate. All isolates were similar in showing maximum growth on media containing ammonium (ammonium sulfate and ammonium nitrate) as a source of nitrogen buffered with calcium carbonate at pH 6.4, but at other concentrations growth was restricted or no sporing occurred. Oknina (125) reported quantitative my­ celium production differences between V, albo-atrum and V. dahliae when grovm on media providing different sources of carbohydrate and nitrogen. Carbohydrate Source; Verticillium is able to utilize carbon ffom several carbohydrate sources, Isaac (8o) re­ ports type M, grooving well on sucrose, dextrose, and maltose media, but poorly on glycerine medium; type D produced max­ imum growth on glycerine medium, but grew poorly on sucrose and dextrose; hyaline variants grew best on maltose medium and fairly well on glycerine medium. Consumption of cellu­ lose by Verticillia is reported by Van Zindern Bakker (174) and Thakur and Norris (167)• Van der Veen (172) states that pigmentation in different strains of V. albo-atrum develops only in the presence of carbohydrates and a plenti­ ful supply of magnesium, and within a definite range of reaction (pH 5 to 8). Roberts (142) suggests that severity of attack in plants is reduced when the carbohydrate content of the plants is diminished. Pruning of leaves of young tomato pliants growing in infested soil resulted in a re­ duced leaf-shoot ratio. Increased resistance in these 30 plants was attributed to reductlomin the carbohydrate content. Increased resistance was indicated by fewer plants becoming infected and diminished severity of the disease in those plants succumbing to infection. Roberts explains this phenomenon by stating that the fungus, even after enter­ ing a plant, is unable to maintain itself unless supplied with sufficient carbohydrate;; it is further suggested that this might explain the effect of shading in controlling the disease in plants already infected as demonstrated, by Bewley Cl^-). Enzyme and Alkaloid Influence; Increased peroxidase activity, due to the action of the fungus (V. albo-atrum) on the oxidizing system of the cell by means of a peroxi­ dase activator in the mycelium, causes lowered resistance in the parenchyma cells adjoining vessels invaded by the parasite. (guchorukoff and Strogonoff, l60). Ovcharov C128) isolated an enzyme, present in pure cultures of V. albo-atrum, Botrytis cinerea, and Fythium de Baryanum, capable of splitting off urea from gelatine in the presence of toluol and chloroform. The name "deurease" was suggested for the enzyme which is considered to play an important part in the assimilation of nitrogen

from protein by the fungi. According to this report^ the greater part of the enzyme was retained in the mycelium as an endoenzyme and the rest was excreted into the medium. 31 Fermentative activities for various enzymes by Y. albo- atrum and V. dahliae were noted by Olenina (12^). Greathouse and Bigler C6 7 ) have shown V. albo-atrum, grovm in liquid culture, to be quite tolerant to a series of alkaloids including sanguinarine, delphinine, berberine, gramine, solanine, and veratrine.

Effect of Dyes s.- Verona (I7 8 ) reports that the growth of four Verticillium species, V. albo-atrum, V, dahliae, V, amaranti, and V. tracheiphilum, was inhibited by mala­ chite green and brilliant green at concentrations between 1;;200,000 and 1 ;.500,000. Williams et al, (186) noted that cultures of the chrysanthemum wilt organism kept in an aqueous solution of malachite green (1:200,000) for 96 hours were killed, though higher concentrations were tolerated for shorter periods. Cause of Wiltr At least two principal theories have been presented in explanation of the exact cause of wilting and desiccation of the aerial portions of the infected host plant. Thrombi, consisting of mycelial masses, of gum-like substances, or tyloses, are regarded as the cause by numer­ ous workers, while others maintains that toxic substance pro­ duction by the parasite is the correct explanation for the phenomenon. A. summary and discussion of these theories for work reported prior to 1929 is given by Eudolph (1^^).

The toxin theory, generally considered the most plaus­ ible explanation,has gained additional supporters since 32 Rudolph C.lM-f)) (who favored the toxin theory) published his general paper on Verticillium hadromycosis. Gottlieb (6?), working principally with of tomato, demon­ strated wilting of young tomato plants placed in filtrates of various fungi, including Verticillium albo-atrum. His explanation of vascular wilts in general is based upon the conclusion that the toxic substance present disturbs the normal water relations of the plant by reducing the ratio of transpiration and water absorption below that critical level normally maintained by a healthy plant under favorable condi­ tions. The permeability of the host cells was also increased by the toxic substance; however, it was shown that wilted plants may recover, at least temporarily, when placed in dis­ tilled water. The origin of the toxic substance may be as a metabolic product of the fungus or a reaction product of the host, but Gottlieb was unable to determine the source. The toxic substance may be a complex of several com­ pounds varying in filterable size, according to Fulton (55)» who demonstrated that filtrates from three biotypes of Verticillium from raspberry were not toxic unless the fungi were grown at least thirty days in synthetic broth and exposed to continuous light. Another biotype proved to be an exception by producing a toxic filtrate following expos­ ure to light for only thirty hours. Fulton further records that after ultra-filtration the unfilterable portion 33 produced a wilt without discoloration of the vessels while the filterable portion caused blackening of the vessels without causing wilt, Oknina (125)} on the other hand, could detect no toxic substances capable of causing wilt, and believes the biochemical causes of wilt are inherent in the fermentative action of the parasite.

PATHOGENICITY Correlated with Morphologyr Pathogenicity of the causal organism has been correlated with the morphological appearance of the colonies by some workers whereas others have been unable to do so. Wickens observed that a hyaline saltant, characterized by dense, white, aerial my­ celium and completely lacking sclerotia, was only slightly pathogenic to cotton. The parent culture, a microsclerotial type, was definitely pathogenic. Hansen (72) concluded that three biotypes differed in virulence though only a few tests were made. Presley (133) reports that a microsclerotial type was most pathogenic to Stoneville 2B variety of cotton.

The microsclerotial type was compared to fluffy mycelial and appressed mycelial types, and were designated types A, B, and € respectively. Type A caused 50 percent infection, type B, 8 percent, and type C, only 4- percent. The rate of wilting also differed:, type A caused mlting after l4- days, type B after 19 days, while type C required 21 days to produce this condition. 34- V. albo-atrum is distinctly more pathogenic than is V. dahliae, according to Ludbrook (106), who grew cultures on artificial media for periods of 3 to 6 years without altering the pathogenicity of the species. Berkeley (12) also found members of the resting mycelium (V. albo-atrum) group stronger parasites than those of the pseudosclerotial (V. dahliae) group.

Miles (117) reports severe infection produced by a microsclerotial strain of V. albo-atrum fromi Mississippi, while a California strain producing no microsclerotia in culture caused only mild infection. A Canadian microsclero­ tial strain gave negative results in the same test where cotton was used as the host plant. On potted greenhouse tomatoes inoculated with the same strains, the Canadian strain caused much more severe and earlier symptoms than either the Mississippi or California strains, but the latter were much more virulent than the Canadian form on , snapdragons, beets, and California poppy. m general, a direct relationship between virulence and quantity of microsclerotia produced by isolates of the mint-wilt fungus has been observed. The most vi3nilent iso­ lates were those forming dense masses of microsclerotia, though this was not an invariable response. (Nelson, 123). Donandt C3 8 ) could find no evidence of inherent superiority of pathogenicity of sclerotial over asclerotial strains of 35 ,V* albo-atriim, but did observe varying degrees of viru- i lence for herbaceous and for woody plants. Decrease of virulence or temporary loss of virulence by continual culture on agar media has been observed by Nelson Cl23). Often accompanying this phenomenon, has been a disappearance of microsclerotia. Ebss of microsclerotia in continued culture has been reported by Rudolph Cl^ ^ ) 5 Berkeley et al. (12), and Nelson (123). Tompkins and Ark (170) report equal pathogenicity in isolates segregated into two groups, conidial and mycelial, from mono spore cultures. Van der Veen (172) could find no apparent correlation between virulence and capacity for pig­ mentation though the two groups (microsclerotial and dark mycelial) formed every possible transition in coloration

from very dark to hyaline. Ludbrook (106) reported symptoms occurred earlier on eggplant infected with V. albo-atrum than those infected with V. dahliae. However, symptoms were less evident on tomatoes and potatoes with V. dahliae than with V. albo-

atrum. Temperature Influence;. The influence of temperature upon pathogenicity remains in a doubtful category. Whether

temperature primarily affects the pathogenic capacity of

the fungus or the resistance of host is yet to be deter­

mined by additional investigation. McKeen (111) concludes I 36 that the temperature factor influences the degree of disease incidence primarily through its effect on the fungus. Isaac (80) has shoivn that a dark mycelial type caused wilting of tomato plants only at 21.^ degrees C, and when plants were subjected to higher temperature, recovery from wilt was effected. Tomato plants inoculated with a microsclerotial type and grown at 21.? degrees C., 2? degrees C., and 2? de­ grees C, showed wilt symptoms, but when placed in 29 degrees C. temperature, wilted plants recovered; when replaced in their original lower temperatures, the plants wilted and died. Fulton (,??) relates that microsclerotial and fluffy mycelial isolates from raspberry infected that host at 24- degrees C. but that an appressed mycelial isolate did not infect until 28 degrees C. was maintained. Symptomatic expression of pathogenicity is determined not only by the fungus strain (and origin), but also by the host reactions as well as the numerous environmental fac­ tors to which the host and parasite are subjected. Cross-inoculâtion Experiments; Cross-inoculation-path- ogenicity experiments have been conducted with varying re­ sults. Williams et al. (l88> inoculated Ailsa Craig and Riverside varieties of tomato plants with different strains

of V. dahliae and V. albo-atrum. Riverside variety appeared

to be less resistant than Ailsa Craig, but when the experi­ ment was repeated under apparently identical conditions, the

reverse obtained. It was concluded, therefore, that definite 37 control of factors such as temperature and humidity were necessary before reliable results could be obtained by this method. Dimock (3 6 ) conducted limited cross-inoculation experiments with strains of V. albo-atrum isolated from a number of ornamental plants. He failed to find any host

specificity, and expressed the opinion that isolates capable of infecting any one of the ornamentals.involved in the experiment would also, under the proper conditions, prove infectious to most of the others. Schaible et al. (152) reported isolates of V, albo-atrum from tomato and egg­ plant in Utah and from tomato in California were equally pathogenic on tomato.

It is possible that strains of Verticillium are host-

selective or that certain hosts are susceptible to certain

strains or isolates of the pathogen but are resistant or

tolerant to others. Inoculation experiments conducted by

Snyder et al. (,l59) demonstrated that isolates from cabbage and radish were cross-infective, but those from Brussels

sprouts, cabbage, stock, radish, and nightshade were non-

pa tho genic to Bonny Best tomato in greenhouse tests.

Isolates from olive and blackberry caused severe wilt in

this tomato variety.

An isolate from Gossvpium barbadense was pathogenic to

only eggplant of nine plants used in host-range tests (Cheo,

2 9 ). Ten strains of V. albo-atrum collected from different

hosts and localities were inoculated into Irish Cobbler I 38 potatoes by Mujica (120); two of these, from and egg­ plant, were definitely pathogenic; four strains, from Koel- renteria panicnlata. Chrysanthemum sp., Acer nlatanoides. and , were weekly pathogenic; and four strains from Acer nennsylvanicum, Lonicera sp., Physalis sp., and

Platanus sp., were not pathogenic to the test plant. Baines

(.8) inoculated chrysanthemum, pepper, and pimiento plants with isolates from Viburnum lantana. chrysanthemum, and

with completely negative results. The same iso­ lates all produced hadromycosis in eggplant although the

peppermint isolate appeared weakly pathogenic.

When various plants were inoculated by the stem-wound method with hyaline and sclerotial strains of a cotton iso­

late, eggplant (var. Bl. Beauty) was susce|tible to both

strains; sunflower, tomato (Marglobe), and|Sunn Hemp were

susceptible to the sclerotial strain only, while chili pep­

pers were apparently immune to both strains (^A).

Nelson (121) planted ten highly susceptible hosts of

V. albo-atrum in a field heavily infested with the mint wilt

strain of Verticillium. None of these plants, including

American cotton, Egyptian cotton, okra, eggplant, pepper,

chrysanthemum, raspberry, tomato. Salvia. and Coleus,

showed any symptoms of infection, while all of the test

plants of piperita were killed in the same plot,

A similar test, conducted in a greenhouse, gave results 39 very comparable to those obtained in the field test. Con-^ versely, isolates of V, albo-atrum from cotton, pepper, okra, eggplant, snapdragon, chrysanthemum, blackberry, maple, and other very susceptible hosts did not infect pep­ permint ünder the most favorable environmental conditions for wilt. Nelson (121) concludes from this and other work that the specific pathogenicity of the mint fungus for plants in the genus Mentha and very closely related genera indicates the existence of dissimilar strains within the V, dahliae group. Green (68) had slightly different results in soil inoculation tests with a mint-wilt strain (B IIB)• Disease developed in one of twenty-one weeds tested; eggplants and four of twenty chili plants gave positive results in a test including nine economic species. In cross-inoculation tests with isolates from nine hosts, only two, from tomato and from radish, infected Mentha piperita. Isaac and Keyworth (81) tested isolates from ’’fluctuat­ ing” and "progressive” outbreaks of wilt of hops. All strains collected from progressive outbreaks caused severe wilt of inoculated hop plants while those isolates from fluctuating outbreaks produced little or no wilt in inocu­ lated plants. These results indicated to them that the chief distinction between the two types of outbreaks lies in the strain of V. albo-atrum involved, although soil factors may, perhaps, be partly responsible for differences in the 40 severity of outbreaks. Isolates of Verticillium from Liatris sp., soft maple, sugar maple, Norway maple, American elm, Japanese barberry, and potato, most of which were successfully inoculated into chrysanthemum, eggplant, and Cineraria by Tilford and

Runnels (l68), showed some variability in virulence but all tested were pathogenic.

Additional information regarding cross-inoculation experiments is available by referring to Rudolph’s ( 14-4-) tables and discussion of interhost relationships of Verti­ cillium sp.

MODE OF ATTACK It is generally accepted that the pathogen enters the host plants through the plant root system. The ability of the fungus to penetrate healthy unwounded roots was demon­ strated by Reinke and Berthold (I36). CTther investigators have confirmed this finding as indicated by Rudolph (14-4-).

Isaac C79)> working with sainfoin, showed that V. dahliae can penetrate seedlings through unwounded roots as well as through ruptures from the emergence of lateral rootlets. Three infection channels through roots are described by Nelson (.123) for the Verticillium causing wilt of mint;

(a) piercing the cell walls of root hairs and other proto-

dermal cells; (b) direct penetration of the surface cells at any point along young roots; and (c) through wounds made "by the emergence of adventitious roots from the cor­ tical tissue, or wounds made by insects and tillage imp­ lements. Attack through wounded roots of chrysanthemum was definitely demonstrated by Huber and Jones (78).

Subsequent systemic invasion of the host plant is by way of the xylem according Van der Meer (112), Nelson (123),

Tilford and Runnels (l68), Huber and Jones (18), Garrett

(56)-, and others. Growth of the fungus in the plant is longitudinal and confined to the vascular system until the host is moribund according to Roberts (1^1). Van der Meer

(112) and heyendecker (101) report isolation of the fungus from all vegetative parts of diseased cotton plants. The uppermost vegetative parts of fresh material are reported by Rada (11) to be most productive of the fungus in cul­ ture isolation studies. Both upward and downward longitud­ inal growth have been reported by Van der Meer (112) and

Dimock (35)»

TRANSMISSION AND SPREAD By Seedt The facts concerning transmission and spread of Verticillium are as yet apparently incomplete despite numerous explanations offered by writers on this subject.

The possibility of seed transmission of the disease was

suggested by Taubenhaus (I63;l64-) who cultured iVfO cotton

seeds; from diseased plants and recovered Verticillium from the interior of 119, or 8.3 percent of those seeds cultured 42 in 1 9 3 6 , and from 2.3 percent of 16OO seeds in 1937. Ear­ lier, in 1 9 3 3 , Richardson (I3 8 ) obtained the fungus from all parts of eggplant plants, including the interior of seed aseptically removed from diseased fruit. Seed from 8 fruits were cultured but positive recovery occurred in 6 cases only. Kadow (85) obtained the fungus from within toma­ to and eggplant seeds. Sudden and widespread occurrence of the wilt in Greece caused Sarejanni (l5l) to suspect seed as the transmitting agent. Hansford (.73) stated that Verticil- lium wilt was distributed all over Uganda in seed obtained from diseased plants, but he did not prove this point with experimental evidence. Brown (21) noted that field obser­ vations strongly supported the possibility of seed-transmis­ sion of the disease since plants attacked were usually scattered, one or two in a place, new land in crop for the first time was affected, and the disease in cotton occurred simultaneously in physiographically isolated areas. In a later report (3) the same author reported tracing discolor­ ed vascular strands from the roots of cotton plants to the bolls. Bardueci and Rada (11) thought it entirely possible that fungus hyphae might reach the seeds in diseased cotton plants since it was possible to culture the organism from practically all parts of the plant including leaf petioles and boll peduncles. In 1938, Brown (20) reported isolating the causal organism from the lint on seeds from infested plants but never from acid-delinted seeds. Llosa (105) ^3 plaims to have isolated a fungus of the Verticillium type from the interior of seeds taken from late and immature bolls from seriously infected plants, and concluded that the dis­ ease may be transmitted by seeds. Vascular diseases of other hosts caused by Fusarium spp, have been reported as seed-borne by Snyder (1^8), Taubenhaus (16.2), and Leach ($6). Eudolph and Harrison Cl^9) report several species of Fusarium occurring in healthy cotton seed as innocuous concomitants. On the other hand, Miles (116) thought it more reason­ able that the Verticilli'om reported by Sarejanni was present in Greece prior to importation of American cotton seed, and suggested that the American varieties of cotton perhaps were particularly susceptible to the Grecian strain of Verticillium. Rudolph and Harrison (1^8) concluded after extensive research that seed produced by diseased cotton plants were neither in­ ternally nor externally infected by Verticillium. In a simi­ lar paper, Rudolph (.1^6> reported only two tomato seeds of more than 26,768 cultured produced Verticillium. Rudolph was not convinced that the two tomato seeds were infected even though the fungus had been recovered, but stated that the fungus may have been confined to the funiculus embedded in

the gelatinous matrix that surrounds each tomato seed, Williams et al. (187) was unable to detect the transmission of Verticillium wilt by tomato seeds but recommends, never- theless, that seed-saving for future plantings be made from healthy plants only. Wickens (4^;46) reports that experimen­ tal results were strongly against the possibility of seed transmission of Verticillium wilt of cotton, and that seed lots from severely diseased cotton plants, both naturally and artificially infected, gave rise to healthy and vigor­ ous plants. Several thousand cotton seed from wilted plants were planted in steamed soil in the greenhouse by Presley (133)) but not a single plant developed any symptoms of wilt. Other workers who have rejected the possibility of seed trans­ mission of the disease for several of the known hosts in­ clude: Cabral (.23)) Cheo (29) > Hansford (7*+)) Guba (69), Herbert and Hubbard (77)) and Thompkins and Ark (170), In efforts to explain the sudden occurrence of the disease in isolated or previously uncropped areas, indigenous occurrence of the pathogen in soils has been suggested by Hansford (7^) and Presley (133). By Root Contact; Opinions differ also regarding trans­ mission by root contact. McEay (110) has reported spread from one potato plant to another in the row during the grow­ ing season, apparently taking place underground through con­ tact of the root systems. Garrett (57) states that prospects for control of Verticillium wilt of cotton are poor because its mode of spreading is typical of a vascular parasite, the transmission from diseased to healthy roots being by root contact although an Infected plant does not become infectious > 5 to an adjacent plant until it is near death or dead when the fungus develops on the outside of the root. Isaac C79) noted wilt of sainfoin spreading from one plant to adja­ cent plants. Slight symptoms of Verticillium wilt of plum occurring in the leaves at the extremity of one branch on the side towards a severely diseased tree suggested to Curzi (3 2 ) transmission by root contact. McKay CHO) suggested that roguing of diseased plants might increase chances of spread rather than decrease them because roots would be broken off and left in the ground where the roots of adjoining plants would grow more freely in the absence of the rogued plant. Wickens (46), however, observed a tendency for aggregation of the disease of cotton into areas of relatively high incidence close to sites where disease occurred in the previous season, but without any neighbor-to-neighbor spread. In addition, he found no evi­ dence that roguing affected the total amount of disease or rate of development. By Vegetative Propagation; Plant reproduction by vegetative means has been shown as a method of spreading Verticillium wilt of chrysanthemums by Huber and Jones (78) and Tilford and Runnels (I6 9 ). Seed pieces are capable of spreading the disease in potatoes according to Neilsen (124). Successful transmission of the disease by bud grafts on has been demonstrated by Dimock (35)• By Plant Debris; Increase of the disease in the field 46 Is th.o'ugh.t to be a direct result of the common practice of leaving diseased plant debris in the soil where it becomes a source of inoculum for later crops of susceptible plants. Leyendecker (99) was able to isolate the fungus from dry, diseased cotton stalks over a six-month period from Octo­ ber, 1946, to June, 1947, during which time a minimum air temperature of -21 degrees C. was recorded. Blank and Leyendecker (7) demonstrated the potentialities of wilt- infected stalks as a means of spreading wilt, Diseased stalk pieces were applied and turned under in presumably wilt-free soil. A high percentage of Infection was observed in the cotton crop produced on the test plots. It was fur­ ther pointed out by Leyendecker (101), that ideal condi­ tions prevail for the establishment of the fungus in the soil in the Southwest as fields are commonly irrigated soon after plowing. In South African plots from which cotton plant debris was removed there was less wilt the following season than in plots on which debris remained, or to which more debris was added (Wickens, 47). Keyworth (86) per­ formed experiments to show that leaves and stems of hops were important agents of spread of Verticillium wilt of that crop in England. Old potato stalks left in the field carried the fungus over winter but not over two winters according to McKay (110). Roberts (141) hastened the spread of the disease in tomato by bark-ringing infected 1+7 plants adjacent to healthy plants. High inoculum potentials occurring in the upper soil layers are probably accounted for by the incorporation of infected plant material on which microsclerotia may form in great abundance. These micro- sclerotia, in arid regions, probably lie quiescent and viable for long periods of time inasmuch as they resist dry­ ing effectively and can withstand temperatures as high as 120 degrees F, for several months (Wilhelm, 180). By Cultural Practices: Certain cultural practices may influence the spread of the disease either directly by themselves, or indirectly in conjunction with other factors. A number of these practices have been mentioned in con­ nection with other phases elsewhere in this review, includ­ ing;: roguing of plants (McICay, 110), (Garrett, 56)f impor­ tation of infected host plants or plant parts (Herbert and Hubbard, 77), (Barejanni, 15D , (Kadow, 85), (Llosa, 105), (Taubenhaus, 163); improper irrigation practices (Schneider, 153), (Leyendecker, 101) ; tillage practices with resultant root injury (Nelson, 123); continuous cropping of suscep­ tible varieties (Wilhelm and Thomas, 185), (Rudolph, lM+); seedbed preparation (Leyendecker, 100); and soil amendment addition (Wilhelm, 182) , (McKeen, 111), and (Keyworth and Hewitt, 90). Cross cultivation has been shown effective in spreading wilt of hop, by means of debris, over larger areas than one-way cultivation (Keyworth, 86). 48 By Mtsceilaneous Means ; Aerial transport of conidia of the causal organism was considered by Sarejanni (151) to be a possible explanation for the rapid spread of the dis­ ease in Greece. Leyendecker (101) stated that under certain conditions the disease may be spread by spores which are pro­ duced by the fungus on the base of dead plant stems at ground level. Wind and water are suggested as the spreading agents. McKay (110) also had found fruiting growth of the pathogen on old potato stalks at ground level. Dissem­ ination of microsclerotia with dust has been suggested by Wilhelm (180) to account for infestation of lands not pre­ viously having a history of a susceptible crop. This same author (Wilhelm, 180) found that Verticillium was able to persist, in central and southern parts of California, for several years (in one case, 8 years) in soil cropped con- tinously with non-susceptible grain crops, but considered it highly questionable that Verticillium could make indepen­ dent growth in the soil.

ENVIRONMENTAL FACTORS AFFECTING DISEASE Sporadic occurrence of Verticillium wilt indicates that an interaction of environmental factors, as well as requi­ site conditions for the pathogen and host, determines the occurrence and severity of the disease. GMumann (59, p. 25), referring to the environmental conditions for in­ fection states; k-9 “The value of a factor will change as a ^result of interaction with all the other factors, external factors being, to a certain extent, interchangeable."

It is evident from the variety of reports concerning the influence of individual environmental conditions that such an interaction is responsible for a great number of the conflicting statements and claims revealed in the lit­ erature pertaining to Verticillium wilt. McKeen (111) studied the causes of an epidemic (epiphytotic) of Verti­ cillium wilt occurring in the Niagara Peninsula, Ontario, in 1 9 4 0 . At the conclusion of his work, he stated that it is apparent that the epidemiology of Verticillium wilt is not a simple function of appropriate soil moisture and soil temperature for fungus activity, but is a complex and re­ quires much further analysis for its elucidation. By subjecting healthy young tomato plants to varia­ tions in environment only before inoculation and then main­ taining a constant and optimum set of conditions for wilt development after inoculation, Foster and Walker C?4) stud­ ied the effect of environmental factors on disease develop­ ment through action upon the host alone. The organism used in this study was Fusarium oxysnorum f, lycopersici (Sacc.) S. & H., a vascular parasite similar to Verticillium in many respects. Factors favorably predisposing tomato plants to wilt were:? soil or air temperature near the opti­ mum for plant growth, low soil moisture, short day-length, 50 low light intensity, nutrient being low in nitrogen or phos­ phorus, or high in potassium, and nutrient low in pH, Poten­ tial susceptibility of the plants was found to be decreased and consequently their resistance increased by soil or air temperatures above and below optimum for plant growth, by very wet soil, by long daylight periods, and by high light intensity. Increased susceptibility was also conditioned in plants grown in solutions high in phosphorus, low in po­ tassium, high in nitrogen, and also in solutions with a high pH,

SQIL The soil in which the host and pathogen come into close contact with each other may be an important member of the complex, Garrett C57) maintains that soil conditions can only influence the progress of the disease indirectly by influencing the susceptibility of the host plant. Only at the point of root contact do soil factors influence the spread of the disease. Unfavorable soil factors therefore exert much less direct influence on fungi spreading along the outside of plant roots, Garrett (57) further states that it appears that the majority of fungus diseases which are soil-borne are favored by soils of light texture. How­ ever, Cabral (23) notes that Verticillium wilt of cotton, of recent occurrence in Mozambique, is prevalent on the heavy clayey alluvial soils of that region. In Alabama,

Smith and Wilson (157) report the disease confined to a 51 soil series (Decatur) characterized as a clay loam, brown­ ish-red in color, and relatively high in exchangeable cal­ cium content. Miles and Persons (1 1 8 ) observed that heav­ ier sedimentary and alluvial soils were more favorable to the disease on cottons than were lighter, sandier types. Bewley (1 ^) considers clay soils favorable to the develop­ ment of the disease, in tomatoes. Clay as well as sediment­ ary soils are favorable for the propagation of the fungus according to van der Lek (98). Isaac (87) reports average wilt severity of wide variance on two contrasted soil types. Cotton wilt caused by Verticillium albo-atrum is most prev­ alent in low-lying, heavily irrigated soils in Argentina (Fawcett, 50). Evidence obtained in Uganda indicated that the disease increases in intensity near the bases of eroded slopes and declines where cotton is grown year after year in the same plot C*+3) • Leyendecker et al, (.103) mention the occurrence of the disease in cotton grown on heavy,Gila- clay, adobe soil, Rudolph (iM^) observed that the disease in California occurs in severe epidemic form on soils vary­ ing from light sandy loam to heavy clay and adobe types. Other writers listed by Rudolph (lM+) have indicated that they regard loams and sandy loams as favorable to the dis­ ease. In Arizona, Verticillium wilt of cotton commonly occurs on the heavier types of soil and less frequently on

the lighter, sandier types. 52 SOIL MOISTURE Soil moisture is considered by McKeen (111) to be ex­ tremely important in favoring or inhibiting the develop­ ment of the disease. He believes that an epidemic out­ break of Verticillium is contingent upon a high level of soil moisture maintained uniformly throughout the growing season, even before soil temperatures are high enough to permit disease development. It was not determined whether infection occurs abundantly in the early part of the sea­ son and remains abortive until higher temperatures prevail, or whether high soil moisture in the early part of the sea­ son merely activates the fungus in the soil, with infection being initiated only later when the soil becomes warm. Van der Meer (112) showed experimentally that wilt of potato was most severe when grown under very dry conditions. Ru­ dolph (144) reports wilt occurring in severe proportions on both very wet and very dry soils. Variation of the soil moisture had no great effect on severity of the disease in eggplant (Ludbrook, 106). Schneider (153) reported that in irrigated plots of guayule the numbers of infections were inversely proportional to the frequency of irrigation, and that diseased plants did not recover as well in dry plots as in those that were irrigated. Williams et al. (189) presents experimental evidence indicating that infection of Potentate variety tomato plants by V. albo-atrum was checked 53 by warm, moist conditions and that by V. dahliae somewhat less. High soil moisture delayed the onset of attack by V. albo-atrum. but the final intensity of the disease was not reduced. Leyendecker (101) recommends reducing soil mois­ ture to minimum levels to reduce the severity of the dis­ ease on cotton in New Mexico. Damage in heavily infested areas has been held to a minimum by keeping soil as dry as is commensurate with desirable plant growth. As indicated by Rudolph Cl^^)? most writers regard drought as distinctly favorable to the disease, but opinions are definitely varied.

SOIL AMD AIR TEMPERATURE

Attempts have been made to show a direct relationship between temperature and disease occurrence in the field. Gratz (6 6 ), planting replicated plots of infected potato tubers in Maine and Florida simultaneously, observed that a high percent of wilt occurred in Maine and practically none in Florida. Similar temperatures occurred during the respective growing seasons in the two localities; therefore, losses should have occurred in Florida, but over six year’s observations this did not take place, Shapovalov and Les­ ley (15^)' experimented with fungus wilts of tomato and ob­ served that, as a rule, Fusarium was more frequent during the hotter part of the growing season, whereas Verticillium tended to gain the ascendency with the advance of cooler weather, not infrequently superseding Fusarium in the same 54 plants. Sugar beets, growing in infested soil, wilted at an air temperature of 75 degrees P.,, while a similar lot, growing at 60 degree's P. for the same period, showed no wilting at all (Gaskill and Kreutzer, 58). With guayule in the San Joaquin Valley, Schneider (153) correlated positive wilt with mean air temperatures of 70 degrees P. Wo nevf infections were noted when mean air temperatures were about 80 degrees P. Eastham (42) has stated that incidence of wilt in tomato is closely related to temperatures of soil and air: when low tempera­ tures prevail, sudden wilting and premature death occur; when the temperature is moderately high there is little wilting of foliage; at high temperature the attack slowly abates. Richardson (138) reports wilting of eggplant at soil temperatures of 11 degrees C. and 30 degrees C ., but not at 35 degrees C. Patel et al (130), reporting wilt of eggplant in India, state that infection invariably takes place only in the cool season. According to this report, temperature is the most important factor controlling infection and progress of the disease. Verticillium wilt of mint, according to Kelson’s (123) field observa­ tions, develops most rapidly and severely in seasons of h i ^ temperature and low moisture. Leyendecker (100) associated severity of disease in cotton with soil tempera­ ture, following field tests where the different types of ' ... . - - seedbeds were used. Raised beds and wider spacing of rows, resulting in increased mean soil temperature decreased 55 the Incidence of disease. Verticillium wilt of sugar beets was found to be directly correlated with soil temperatures in Colorado (30), Verticillium wilt was originally believed to occur primarily in cool regions (Rudolph,144). The wide geo­ graphic al distribution of this disease indicates that it is present in a multiplicity of hosts under widely varying climatic conditions in which soil and air temperature may be under varying sets of circumstances, a limiting factor,

SOIL ;REACTION The literature reveals no strict correlation between occurrence and severity of Verticillium wilt and the nature of soil reaction. Taubenhaus et al. (165) associated Verti­ cillium wilt of cotton, then known as Waxahachie wilt, with alkaline soils because, it appeared significant that Texas root rot and Verticillium wilt occurred together in the same fields, whereas Fusarium wilt was never found in. the same areas as Texas root rot. Wilt of eggplant, demonstra­ ted by Martin (109), in confirmation of Haensler’s (71) work; was much more severe in limed plots than in acidified plots. Tehon and Jacobs (166) noted that Verticillium wilt of elm occurred, almost without exception, in soils very close to pH 8.0. In only one case where variance occurred, the pH was exactly 7.0. Occurrence of wilt of cotton has been reported in 56 Alabama as being confined apparently to a soil series having a pH range 6 f 5*0 to 6.3 in which the average pH of the surface soil is 5*5 and the average for the subsoil is 5A. Wilhelm Cl8l) points out that severe outbreaks of

Yerticillium wilt occur repeatedly on soils well in the acid pHi range, and concludes that the occurrence and sever­ ity of the disease is not greatly affected by soil reaction within the range in which susceptible crops are commonly grown.

SOIL FERTILITY Fertility of the soil most certainly affects the character of growth of the crop. It is likely, therefore, that soil fertility and host nutrition affect the occurrence and severity of Verticillium wilt. Evidence obtained in

Uganda indicated that cotton wilt attributable to Verti­

cillium dahliae and Fusarium spp. is more prevalent on fer­ tile than on inf ertile soils C^3). Keyiforth and Hewitt (90)

described experimental work in which Verticillium wilt of hops ( lupulus) was reduced by reduction of nitrogen, phosphorus, potassium or magnesium content of the soil.

Both disease incidence and severity were measured by vis­

ible symptom expression. The pathogen was frequently iso­

lated from symptomless plants. These workers state that

since all the treatments resulted in some degree of plant

starvation, the resultant reduction in disease may possi­ 57 bly be related, more to general starvation than to lack of any one element, bat the evidence on this point was incon­ clusive as the effects observed were not repeated in a second experiment. Elimination of calcium or addition of nitrog en or pho sphoras did not apparently affect wilt in­ cidence or severity. liarge quantities of potassium added to the test soil resulted in a noticeable reduction of the disease occurrence.

Soil amendments rich in nitrogen were consistently effective in bringing about a substantial reduction in the inoculum potential of V. albo-atrum. according to Wilhelm

CI8 2 ). A-. longer decomposition period of some of the mater­ ial s generally gave a larger reduction i n ,inoculum potent­ ial than did shorter periods. Donandt (3 8 ) showed that in plants receiving insufficient nitrogen the parasite is, restricted to the stem bases. With a moderate nitrogen supply penetration of the tops may be effected, but typical symptoms of infection are not apparent. Plentiful applica­ tions of nitrogen, on the other hand, lead to vascular ob- straction, wilting, yellowing, and premature decay. Van der

Veen (172) reported the incidence of V. albo-atrum on toma- toes was: greater where soil was rich in nitrogen. Haensler

(7 1 )! stated that addition of heavy green manure to infested soil aided in the control of wilt of eggplant, McKeen (111) found that addition of green plant residue or of tartaric or citric acid to the soil slightly lowered the activity of the fungus. 58 SOIL MICROELORA

The accompanying organisms are an important part of the environment of any soil fungus. McKeen (111) asserts that the growth and survival of mycelium and microsclerotia of V. albo-atrum is determined to some extent by the competitive effects of other organisms which are also in part a function of moisture and temperature. Competitions may thus be a factor in influencing the aggressiveness of the fungus in any given soil. Growth of the fungus is more rapid in sterilized soil than in natural soil (Huber and Jones, 78).

In sterilized soil Verticillium grew readily but was killed or checked when other organisms including Gliocladium. Chae- tomium. Stachybotrys. and Myrothecium spp. were introduced singly, while Trichoderma. Fusarium. and Mucor spp. pro­ duced little or no effect (Wilhelm, I8 3 ):. Roberts (1^1) recommends the use of steam-sterilized soil for soil inocu­ lation since plant infection is favored. If left for recol­ onization by other micro-organisms before inoculation with

Verticillium. the steamed soil becomes progressively less favorable for infection by the fungus.

In m i t -inf est ed soil the incorporation of a ciliated protozoan, Culpoda. materially lowered the incidence of dis­ eased plants and substantially increased yields of tomato plants (Brodski, 17). Verner et al. (177) discovered that normal soil introduced into sterile soil ten days after in­ oculation of the sterile soil with V. dahliae caused the 59 g3?bwth of the fungus to dec Tine and f lhally stop altogether-, Kononenko (94) reports' that most of the soils examined in

Aimtenia- ( H. 8 .8 ,R. ) were found to cbntain myfxohacteria( . (PolyangiUm and Myxocbccus'spp.) which are capable of causing lysis bf the mycelium .'of V. dahliae in laboratory culture8 and of hindering sclerotia1 development and dès- - trbyihg young mycelium in the soil. Mycolytic bacteria, particularly those attacking V, dahliae, develop well un- ' der alfalfa and clover, but not at all under cotton and flax (Krassilnikov, 95). Studies of V, albo-atrum, made by Wilhelm (183), indicate that it is a soil invader father than â soil inhabitant. The difference, as defined by the author, is that an invader has a saprophytic exis­ tence restricted to a precarious tenancy of plant material

invaded while pathogenic; a soil inhabitant grows sapro­

phytic a 11 y in competition with general soil microflora.

In sterilized soil Verticillium grew readily but in com­

petition with other organisms did not grow through the soil nor did it colonize a ring of tomato stem-pieces 1 cm. distant from an inoculum stem-piece in 6 weeks. Tomato can bring about a rapid increase of Verticillium in the soil, but was not found as a component of. the root surface of rhizosphere microflora of tomato,

CROP HISTORY The crop history of the soil has been considered as a 60 factor influencing the incidence and severity of Verticillium wilt. Numerous investigators have made recommendations for crop rotation, a review of which is given by Eudolph ( i W ) . Potato and tomato crops are referred to most frequently in connection with build-up of Verticillium in the soil. Wilhelm and Thomas (.185^) have shown progressive increase of infection of tomato plants in successive plantings on the same soil. June (8 ^) cautions that stone fruits should not be grown after tomatoes or potatoes for at least three or four years.

VERTICAL DISTRIBUTION OF PATHOGEN In determining the vertical distribution of V. albo- atrum . Wilhelm ClBO) detected the fungus in soil from 30-36 inch depths. The 0--6 and 6-12 inch depths were found to contain 3 to times the degree of infestation of the deeper soil layers. No relation was observed between the type of soil or crop history of the land, including depth of root penetration, and the vertical distribution of the pathogen.

CONTROL OF THE DISEASE

RESISTANT VARIETIES A completely practical and effective means of control has not been found for Verticillium wilt. Breeding and se­ lection of wilt-resistant varieties in certain crops is the only means holding promise for use in the widely varying 61 situations in which the disease occurs. Rudolph and Harri­ son Cl4-7) successfully selected resistant strains with the cotton varieties Cooke 307-6, Mexican Big Boll, Kekchi, Tuxtla, and Missdel. Strains of Stoneville and Acala, while not resistant, were prolific under heavy infection. American-Bgyptiàn types were found highly resistant to the causal organism. The New Mexico Cotton Field Station developed a new cotton variety, Acala; 1^17 WR, which was resistant to Verticillium wilt. This variety was selected for release in 194-5 (137) • Leyendecker (101) recommends the use of American-Egyptian varieties Fima 32, Amsak, and SxP in the Rio Grande Valley since they are less susceptible than Upland varieties. Of the last named, 1517 WR, Mesilla Acala, and l5l7 B are reported showing some tolerance to the disease. Eresley (133)) notes that Upland cottons (G. hirsutum) appear susceptible to Verticillium wilt and that Egyptian, Pima, Sea Tsland, and South American cottons (G. barbadense) have a high degree of resistance or tolerance to the disease, but when resistance tests are performed in different localities the degree of resistance apparently varies. He theorizes that the change may be due to the effect of the environment on the host, on the pathogen, or to genetic differences in the pathogen, Herbert and Hubbard (77) tested Acala, Mebane, Delfos, and Pima varieties for susceptibility and found that none 62 showed resistance except Pima, of which most plants were diseased though they showed no conspicuous external symp­ toms. Resistance to the disease has been shown to be gen­ erally associated with late maturity, small bolls, and short staple according to Harrison (76). The selection of a wilt-resistant variety of Gossypium barbadense L.. called "Tanguis", has been accomplished in Peru, where some degree of tolerance has also been noted in Giza 7 introduced from Egypt and to a lesser extent in Sakellaridis, Pima, Sea Is­ land, Semiaspero, and Rihon. (Barducci, 10). Sea Island cotton developed infection in Argentina, according to Drummond (39). Anatomical studies of cotton have been made by Sukhor- ukoff Cl6l). with a view to selecting resistant varieties on the basis of anatomical distinctions. According to this report, immune varieties have a firm pith of small cells and medullary fays of many layers always filled with reserve starch whereas the opposite is true for susceptible varieties, Fedotova (.51) has developed a serological method of determin­ ing the disease resistance of a number of strains of Gossyp­ ium hirsutum, G* herbaceum, and G. barbadense. Gerstel (60) hints that resistance in guayule may be related to chromosome number since there was increasing re­ sistance in diploids, triploids and tetraploids, respectively. Kelson Cl22) has developed resistant varieties in spearmint by breeding and selection. Dimock (3^) has secured virtual 63 immunity from Vertioilli-um wilt in chrysanthemums by shoot- selection from rogued-stock plants and suggests use o,f this method with a number of ornamental plants and bush fruits#

CULTURAL PRACTICES Leyendecker et al, (103) published a preliminary report covering one year of work regarding several cultural prac­ tices holding some promise for the control of Verticillium wilt of cotton in New Mexico. A combination of three prac­ tices including one-year rotation, extra high seedbeds, and thick spacing of plants in the row has been recommended. One-year rotation following barley produced an exceptionally good yield. Double-row seedbeds 1^ inches high decreased the incidence of Verticillium wilt and increased the yield of the cotton crop by increasing the soil temperature of the seedbed. Clumps of plants one foot apart produced higher yields than single plants one foot apart and disease inci­ dence was lower in the clumped plants than in those occur- ïîing singly in each hill. Dry fallowing of fields to reduce prevalence of wilt and increase yields as well as using no more irrigation water than is necessary for cotton production was recommend­ ed by Leyendecker (100) in an earlier publication. Wilt

increase was noted as the soil moisture wæs increased, while decreased soil moisture, or less irrigation water than normal, decreased the incidence of wilt; however, it also 64- damaged the cotton by drought.

Since wilt-infected cotton stalks may spread the dis­ ease the removal of diseased plants during the growing sea­ son might reduce the spread, but would probably be of value in, fields showing only occasional diseased plants. (Blank and Leyendecker, 1 6 ),, Compulsory prophylactic measures, such as removal of all plant debris, the use of fresh manure, and crop rotation, such as alfalfa after cotton, are recommended by Soloveva and Letov 0-29}. Zeller (.191) has recommended cane-fruit plants be rogued provided incidence of disease is under ^ percent. Three- or four-year rotations with two or three nonsuscept­ ible crops also proved experimentally effective in elimin­ ating the fungus from the soil and is recommended together with the planting of healthy nursery stock. Three potato- plant rpguings instead of one was recommended by McKay CHO) since iiifected plants do not always show symptoms because of late infection. Two-year rotations were found ineffective though three- and four-year rotations were considered effect­ ive in eliminating the fungus. Control methods on tomato recommended by- Eastham (4-2) consisted of using clean seed and seed dusting, raising young plants under sanitary condi­ tions, and six-year rotation. He also mentioned that any cultural practice which would raise the temperature of the environment should reduce losses. 65 SOIL MIENDMENT8

The addition of various soil amendments has not pro­ duced satisfactory and reliable results. Wilhelm (182) states that control of wilt by soil amendment would require approximately 0,075 pounds of actual nitrogen per cubic foot of soil, or more than 1.5 tons per acre foot, and may ex­ plain in part why field attempts by amendment for control have generally been unsuccessful. Some of the substances added to soil for control of the disease without success are: sulfur or lime (Williams, l8y), potassium sulfate and guano (Barducci, 9)j ammonium sulfate

(Rada, 13^)? and aluminum sulfate (Guba, 6 9 ). Garrett (57) has stated that manurial treatment of soil is useless in combating the disease since infection is favored by a high level of nutrition in the host plant. Keyworth and Hitch­

cock (9 1 ) observed that late side-dressing of hop plants in­ fluenced the "forwardness" of the vines and tended to reduce the number of affected plants but not the severity of the symptoms. The proper application of cyananiid to the soil may reduce the amount of the disease in eggplant to a con­ siderable extent (GÜssow, 7 0 ).

CHEMICAL DISINFECTION Control by chemical disinfection has been tried and found effective but in general is so costly that extensive field use is prohibitive. Soil fumigation with CWP-55 66 Ctechnlcal chlorobromopropene) has been tried with progress toward control by Wilhelm Cl8^)• Chloropicrin has been used successfully by Ferguson and Wilhelm (52), Kligman (93), and Godfrey (6l). D^D reduced the amount of disease according to the 23rd Annual Report, New Zealand (33) * Ferguson and Wilhelm (52) also tested allyl bromide and Shell CBP-55 (55^ chlorobromopropene) in 32- gallon cans of soil and found these chemicals diffused laterally and downward but not upward. Eighteen other chemicals including carbon di­ sulfide, ethylene dibromide, formaldehyde, pentachlorethane, propylene bromide, and tetrachlorethane were not effective against Verticillium in these tests. However, Jacks (82) obtained satisfactory results with formaldehyde and para­ formaldehyde .

SEED TREATI4ENT Hot water seed treatments have been recorded las being satisfactory for infected eggplant seed by Osmun (127), for tomato seed by Van Foot and Wiertz (173), and for both egg­ plant and tomato seed by Kadow (85). Brown (19,20) has recommended the use of acid-delinted cotton seed as a pre- caiitionary measure in combating the spread of Verticillium. Moore (119) points out that seed testing is not in itsjolf a particularly effective method of controlling seed-borne dis­ eases in general (presuming that Verticillium wilt of cotton MAY be seed-borne), and in view of the large number of seed-

borne parasites and the many ways in which they can be 67 carried it is often impossible to detect the parasites merely by examining or even germinating the seed. There­ fore certification of healthy seed-crops in the field seems to offer the only solution for controlling spread of a dis­ ease which may be seed-borne.

LEGAL MEASURES. Legal measures have been taken to control spread of Verticillium wilt of hops in England and Wales. Notice in writing to the Ministry of Agriculture, of the existence or suspected existence of the disease is mandatory, as are the destruction of diseased plant parts, and prohibited sale for planting of plants or parts grown on infested land. 68 M E T H C D S A N D R E S Ü L T S

COLLECTION AND DESCRIPTION OF CULTURES

An isolate from cotton gro-wn near Tipton, Oklahoma, was secured through the courtesy of Dr. L. M. BlanJc, College station, Texas, in April 19^9- This isolate, here­ after referred to as the Tipton strain, had been cultured on artificial media for some time prior to coming into the possession of the writer. Xt was characterized by very sparse production of pseudosclerotia in culture. Mycelial growth was appressed, greasy gray-white, and frequently formed fasicular, pointed tufts of aerial mycelium. Con- idiophores and conidia were typically few in number. A second isolate was obtained by Dr. Alice M. Boyle, Department of Plant Pathology, University of Arizona, in June, 1950 from cotton grown on the University Farm, Tucson, by Professor William X. Thomas. It was a pseudosclerotial- plus-mycelial type, quickly forming dense black ciusts of pseudosclerotia and great quantities of fluffy white aerial mycelium. When first observed by the writer, production of conidiophores and conidia was lacking, though the original parent culture had formed these structures abundantly. This isolate is hereafter called the Thomas strain. In September, 1951? a Tërticillium of the dahliae type was cultured from cotton gro-wn near McNeal, Arizona, and has 69 been'named the McNeal strain. This strain was similar to the Thomas strain in gross appearance though forming slightly less fluffy aerial mycelium. Such mycelium as was formed occurred in small, dense, white tufts. Pseudo­ sclerotia were formed in profusion, but conidiophores and conidia were few in number, even in young cultures. A- Verticillium of the albo-atrum type was obtained from the Commonwealth Mycological Institute, Kew, Surrey, England, in July, 1951» as a sub-culture from their iso­ late No. I.M.I. M+578. The parent culture was isolated from hops at Penshurst, Kent, in 19^9 and was supplied to the Commonwealth Mycological Institute by R. V. Harris of East Mailing Research Station at the request of Dr. I. Isaac, who stated that it represented his (dark mycel­ ium). type described in Trans. Brit, mycol. Soc., 32:137- 1^7, 19)4-9 . It is a strain of the “-fluctuating” type and is characterized by the formation of numerous dark-brown, smooth to torulose, thickened, unbudded, resting hyphae. No pseudosclerotia were formed. Rapid growth was usual with abundant production of verticillate conidiophores and typical conidia. Aerial mycelium was moderately suppressed with occasional fluffy tufts of gray-white color. Another Verticillium. hereafter called the Sahuarita strain, was isolated from the pith of wilted cotton plants collected at Sahuarita, Arizona, in August, 1952. It is a pseudosclerotial plus mycelial type identical to the 70

McNeal strain.

K sixth, isolate, hereafter referred to by. its: generic name. Glio cl a dima. was obtained from old cultures of lower stem-portions of cotton plants inoculated with Verticillium. and was mistakenly identified by the writer as a form of Verticillium. Later identifications made by two other agencies proved the fungus to be Gliocladium roseum Bainier*

It formed neither pseudosclerotia rear resting mycelium. Aerial mycelium first appeared in culture as small loose-

floccose white tufts, later becoming buff-yellow and occa­ sionally light salmon to flesh-colored in the fruiting areas.

Conidiophores were vertical, verticillate, and numerous. ! Conidia occurred in Aerostalagmus-type, gelatinous heads on fresh media and were not observed forming chains.. Elongated,

agglutinated heads of conidia of the Clonostachys-type were later observed occurring in very old, dry cultures. Conidia were hyaline, elliptical, and slightly apiculate. Conidio­ phores were of two types:: (a) short, coarse, with phialid- like branches forming compoundly-branched verticels, and

(b) long, slender branches, with few verticels (Fig. 1). On potato-dextrose agar, the fungus causes a definite yellowing

of the substrate. Figure 2 shows typical colonies of Gliocladium and the first four Verticillium isolates described; the colonies are 7 days old, growing on potato-dextrose agar, and were, incu­

bated at 23 dég. C. 71

preliminary : experiments w i t h s x p s e e d s On March 8 j 1950, approximately 150 cotton seeds of the SxP: variety were hypodermically inoculated with the Tipton strain by injection of a sterile-distilled water suspension of conidia and mycelial fragments. Penetration Of the in­ teguments of the seed was facilitated by softening these parts by soaking the seed in sterile distilled water for approximately one and one-half to two hours. The outer sur­ face of* the seeds was disinfected prior to inoculation by immersion in a 1 : 1 0 0 0 solution of mercury bichloride for 3 minutes ; excess disinfectant was removed by two rinses in sterile distilled water. This method will hereafter be called Method A. Inoculum was prepared by maceration of two-weeks-old . colonies grown on potato-dextrose-agar slants. Five ml. of sterile distilled water were added to each slant to form the suspension. Each colony was examined microscopically to in­ sure that numerous conidia were being produced. Inoculum was poured from test tubes to sterile, 50 ml. Ehrlenmeyer flasks from which the hypodermic syringe was easily filled. Injection of the inoculum was made near the micropylar end of each seed. One-tenth ml. usually was sufficient to cause an overflow of inoculum through the micropyle. Inoc­ ulation equipment consisted of a 2 ml. hypodermic syringe (Br-D Yale, No. 2Y) and a 23-gauge hypodermic needle (B-D Yale, No. L.N.) shortened to approximately one-eighth inch. 72 A l X 3 X 8 -inch white-pine board in which holes, one-quar­ ter inch in diameter and one-quarter inch deep, had been drilled^ served to hold seeds firm for inoculation (Fig, 3), Theholding-board was surface-sterilized before, and period­ ically during, use by immersion in 1 : 1 0 0 0 mercuric chloride for at least 5 minutes, A small wad of cotton wool packed into each hole in the board served as a reservoir of liquid mercuric chloride to kill excess inoculum flowing from the inoculated seed. Seeds were transferred from sterile Petri dishes to the hoiding-board and back to other sterile Petri dishes by flamed forceps. The hypodermic syringe and needle were pro­ tected at all times between injections by cotton wool bear­ ing an excess of 70 percent ethyl alcohol and thoroughly rinsed with several changes of sterile distilled water. After:-inoculation moist seeds were placed in sterile Petri dishes for further observation and Use, Kiree days after inoculation approximately half of the seeds showed small white mycelial colonies growing from either the punc­ ture wound or the micropyle, and in some Cases, from both sites. In 10 days the colonies had ceased growing. A por­ tion of the seeds was kept in the sterile dishes at room temperature until October 2 8 , 1950, (Fig, M-) when some of them were cultured on potato-dextrose-agar following a five- minute immersion in Rada's (11) disinfectant of alcoholic-

. i > mercuric chloride (250 cc, of 3 5 percent ethyl alcohol, 2 grams mercuric chloride, and 750 cc. distilled water), 73 ATter seven" days on the agar at 25 degrees C, the fïingns was again produced from within the seeds (Figs, 5 and 6). On December l8, 1950, sixty seeds of this lot were planted in sterile soil in the greenhouse to test the effect of the fungus upon the seeds. All seeds selected for this experiment bore visible post-inoculation Verticillium col­ onies at the micropyle or wound in the seedcoat; 15 seeds were planted in each of four 12-inch clay pots. One pot of 15 unwounded, uninoculated seeds of the SxP variety was similarly planted as a check. None of the inoculated seeds produced seedlings,although 13 of 15 seeds of the check developed normally. At the same time as the previously described experiment, visibly diseased SxP seeds were planted in hills in sterile soil with healthy seeds of the 1517 RB variety. Five 12- inch clay pots contained 3 hills each, each hill consisting of one infested SxP seed and three healthy 1517 RB seeds, A: check pot of 3 hills of 4- healthy 1517 RB seeds was estab­ lished. In l4 days, 31 of *+5 healthy seeds planted with in­ fested seeds had produced seedlings normal in appearance; 11 of 12 check seeds produced normal healthy plants. At 25 days, two seedlings growing from infested seed hills died. Culture of killed seedlings produced only Rhizoctonia sp. All the plants were grown until May 1, 1950, when the plants were removed from the soil and examined for internal symptoms.

Results of the examination were negative. External symptoms 7^ of disease were lacking; test plants and controls, show­ ing no differences. Cultures were made of stem and root pieces of 2? test plants and of 5 control plants. Stem and root pieces 1/2 to 1 inch long were immersed in Rada * s alcoholic mercuric chloride solution for one and one-half minutes, rinsed in sterile distilled water, flamed momen­ tarily, and plated on potato-dextrose agar having a pH of 7,2, Plates were kept at room temperature until June 10, 19^1, and were examined periodically for presence of Verti­ cillium, Gliocladium roseum was found June 9th on 7 lower stem pieces but Verticillium was not recovered.

HISTOLOGICAL STUDIES QF VERTICILLIIM-INOGULATED SEEDS For histological study a number of hypodermically inoc­ ulated SxP seeds, after removal of the integuments, were killed in a formalin-aceto-alcohol solution (Johansen, 83), dehydrated through a tertiary butyl alcohol series, and embedded in paraffin. Longitudinal sections of infested seeds were cut by rotary microtome, mounted, and stained with several stain combinations including safranin-fast green, safranin-light green, and safranin-Heidenhain*s iron h&na- toxylin. Removal of the seed coats from seeds to be killed frequently allowed dry, granular-embryo tissue along the course of penetration of the inoculation needle to fall out, leaving a small, sunken, corky, brown-colored cavity in the 75 side of the naked embryo. Freehand sections Vere made of some of the discarded seedcoats. Intercellular and intracellular growth of the fungus was found in the inner, thin, brown—colored fringe. tissue (remnant of the nucellar sac?). In several instances, two swollen bulb-like portions of a hypha, on opposite ^ sides of cell walls, were seen to be joined by a very thin hyphal portion passing through the cell walls. The exact manner of penetration of cell walls by Verticillium hyphae was not determined. Prepared slides showed Verticillium hyphae generally massed in the spaces of the cotyledenary folds; however, the palisade cells of the cotyledon were penetrated as were the spongy parenchyma cells adjacent to the palisade layer, A few of the parenchyma cells appear to have been partially lysed (Fig. 7). A. camera lucida drawing of penetrated cells is included in the appendix as Figure 8, In those cells where hyphae were seen to occur, the stored reserve materials appeared to be reduced, as indi­ cated by a markedly decreased stain acceptance by the affect­ ed part. Aleurone grains showed signs of disintegration in the immediate proximity of the hyphae. Nuclei in affected cells appeared asteroid in shape, possibly as a result of lytic processes by the pathogen. Nuclear migration as a reaction to cell penetration by the hyphae was not evident. 76 Invasion of uninjured tissue was not general, thereby- indicating that Verticillium is weakly parasitic under such circumstances.

PATHOGENICITY TEST USING VERTICILLIUM-INFESTED OATS INCORPORATED INTO SOIL In order to determine if the five strains of fungi under study were pathogenic to cotton, soil was inocu­ lated with the isolates gro-wn upon whole steamed oats. Inoculum was prepared by placing 2^ grams of whole oats in 7^0-ml.. Ehrlenmeyer flasks to which an equal vol­ ume of distilled water was added to make a loose, individ­ ual-grained medium which would be easily applied and mixed into the soil. The oat medium was steamed in the auto­ clave at 2^^- degrees F. for 20 minutes and cooled to room temperature, at which time water suspensions of the macer­ ated fungus colonies were poured over the prepared oats. In 1^- days at 23 degrees C. the oat medium inoculated with each of the isolates appeared to be thoroughly permeated; - \ however, to insure complete, invasion of the medium the flasks were vigorously shaken to break up and mix the sub­ strate and incubated an additional three days before being used. Flasks of steamed oats to which only sterile dis­ tilled water was added were prepared in a like manner to be used for inoculation of soil in control pots. Five 12-inch pots of steam-sterilized soil were inocu­ lated with each of the fungus isolates at the rate of 3 77 level tablespoons of inoculum per pot. The inoculum was stirred into the soil, which was surface-irrigated and allowed to stand for 7 days before planting. Pots were covered with heavy brown wrapping paper for protection from dust and insects during this interval. Fifteen seeds of the 1517 RB variety were planted in each of the pots. Seeds were surface-sterilized in 1 : 1 0 0 0 mercuric chloride for 1 minute and were handled aseptically until planted. First emergences were observed on the third day. In two weeks emergence was completed and a number of seedlings were observed to be wilted and some were dying. Isolation from wilted or killed seedlings proved success­ ful in 5 of 25 attempts: McNeal strain was recovered in two instances, CMI in one, and Gliocladium in two cases. Pénicillium and Fusarium species occurred repeatedly in these cultures. At 10 weeks a number of surviving plants inoculated with Thomas, McNeal, Tipton, and CMI strains exhibited typi­ cal foliar symptoms of Verticillium wilt (Fig, 9)- Plants growing in control pots and those inoculated with Glioclad­ ium were unaffected. Examination was made of the surviving plants for vascular discoloration. Table h lists the number of seeds planted in each 5 pot series, the emergences, those plants wilted or dead two weeks after planting, and at ten weeks those showing foliar symptoms of wilt, and the number of cases in which vascular discoloration was found. Cultures TABLE 4

Results of pathogenicity test in soil artificially infested with steamed oats inoculum*

At 2, weeks At 10 weeks Strain Planted Emerged Wilted Dead Foliar Vascular ■ symptoms discoloration

McNeal 75 58 26 10 3 "4 ^

Thomas 75 63 . 18 8 2 6 Tipton 75 69 8 10 2 3

CMI 75 73 11 B 6 8 Gliocladium 75 68 4 0 0 0 Control 75 68 0 0 0 0

03 70 made of all plants showing internal symptoms resulted in recovery of McNeal and CMI strains but Thomas and Tipton strains were not reisolated.

LA.BORATORY STUDIES SEED. INOCULATIONS. 1 9 % in March, 1 9 5 0 , additional seed of several cotton varieties were obtained from the Cotton Laboratory, Univer­ sity of Arizona. Seed were from the 19^9 crop and included Pima 3 2 , SxP", 1517 RB, Acala 33, and Paula C. Seed of these five varieties were inoculated with Tipton isolate by the previously described Method A, Following inoculation, seeds were placed on sterile filter paper and allowed to air-dry 12 hours after which time they were surface-sterilized in mercuric chloride for 30 seconds, rinsed twice in sterile distilled water, and again air-dried under three thicknesses of cheesecloth. Care was taken not to agitate the seed in the second sur­ face -sterilization so that the small air bubbles formed at. the inoculation hole and micropyle remained in place. This prevented penetration of the disinfectant into these open­ ings. Seeds were stored at room temperature in small ster­ ile Erhlenmeyer flasks stoppered with sterile cotton wool, A few seeds of each variety were discarded before storage because the radicle had penetrated the micropylar opening during the inoculation process. Table 5 shows the seed 80

TABLE ^ Inoculation of cotton seed "by hypodermic injection.

Cotton Pre-soaking Number Date variety time (Hours) inoculated inoculated

Acala 33 6-7 200 3/22/50 Do Do 500 3/23/50 SxP li-2 600 3/29/50 Do Do 384 4/5/50 Pima 32- 3-M- 500 4/12/50 Do Do 350 4/13/50

1517 BB if-5 400 4/14/50 Paula C 3-4 360 4/20/50 81 variety, pre-soaking time, number of seed inoculated and date of inoculation. Hypodermically inoculated seeds were cultured period­ ically on potato-dextrosé agar to determine the efficiency of inoculation, the effect on germination of the seed and the length of time tha t the fungus r©nains alive in the inoculated seed. Tables 6 and 7 show these results. Figures show only positive identification of Verticillium. Thirty other seeds produced Verticillium-like hyphae without fruiting. Seeds: were surface-sterilized before culturing by immersing them in mercuric chloride (1:1000) or Rada’s disinfectant for periods varying from 30 seconds to 3 min­ utes. Fewer contaminations occurred when Rada’s disinfec­ tant was used for a period of one and one-half to three minutes,although the inoculum probably was also killed in a number of cases due to penetration of the alcoholic solution through the inoculation wound and the micropyle. The lot of Acala 33 seed inoculated by this method was found completely contaminated by Aspergillus sp. and was not used for tube culture tests. Verticillium growth was noted occurring from the wound- site CFig. 10), the micropyle, and chalaza, and most frequent ly from the thin brown fringe tissue (remains of the nucellar sac} when the seed coat was cast off. Growth on and in the cotyledons themselves occurred only in the immediate vicinity 82

TABLE 6 Cultures of hypodermically inoculated seeds.

mm STAPLE v a r i e t i e s :

grodiicihg Verticillium Date ” Numher of Seeds . Net ...... cultured seeds germinated Germinated germinated Total

...... 5/3/50 5 3 1 0 1 5/10/50 5 1 1 2 3 6/8/50 5 h 2 1 3 6/28/50 5 1 1 1 2 10/21/50 2 2 1 0 1 11/V50 5 3 1 0 1 5/28/51 ^ 1 2 h 2 6 Pima 32

5/3/50 5 5 3 0 3 5/10/50 5 5 3 0 3 6/8/50 5 4 2 0 2 6/28/50 5 2 0 0 0 10/21/50 2 2 0 0 0 11/V50 5 ^ 1 0 1 5/28/51 48 25 3 1 4 9/26/51______3______1______O ______1_ TOTALS 155 76 24 ...... 7 31 PERCENT 49.03 20.00 83

TABLE 7 Cultures of hypodermically inoculated seeds. SHORT STAPLE VARIETIES"

Producing Verticillium Date Number of Seeds Not cultured seeds germinated Germinated germinated Total

Paula C variety g/3/^0 5 5 4 0 4 5/10/50 5 5 2 0 2 6/8/50 5 5 2 0 2 6/28/50 5 1 0 0 0 10/21/50 2 2 0 0 0

1 1/ V 5 0 5 5 0 0 0 5/S8/51 48 21 7 3 IG 9/S6/51 5 5 0 0 0 1517 RB variety

5/3/50 5 5 4 0 4 5/10/50 5 5 2 0 2 6/8/50 5 4 0 0 0 6/20/50 5 2 0 0 0 10/21/50 2 2 2 0 2

1 1/V 5 0 5 4 1 0 1 5/28/51 48 37 11 4 15 9/26/51 5 5 1 0 1

TOTALS 160 113 36 7 43 PERCENT 70.62 26.87 84 of the wound-site, although occasionally, the fungus having become established through the wounded tissue, continued to invade the cotyledon. Invasion was evidenced by brown, some­ what watersoaked areas having a sharply delimited advancing margin. Growth of the fungus was slow and often died out entirely leaving only a browned, shrunken lesion in the other­ wise green cotyledon CFlg» 11). The hypocotyls also were attacked in several instances, in which the fungus produced sunken brown lesions which, in some cases, girdled the seed­ ling causing death of the seedling plant. Growth on andiiin the seedling plant was not particularly vigorous; the fringe tissue and the seedcoat provided a much more favorable medium for fungus development. Seed coats were frequently completely invaded by the fungus; the fringe tissue often tore during germination and small portions of this tissue remained attached to the lower epidermis of a cotyledon. These small portions of the fringe tissue sup­ ported growth of the fungus although the cotyledenary tissue was not invaded. It will be noted that the pathogen was able to exist in viable inoculated seeds for over 17 months after inocu­ lation while stored under approximately normal conditions.

Bacterial contaminations resulting fromuhandling materially reduced the germination rate, especially in the SxP and Paula C varieties. The long-staple varieties also appeared less able to withstand the injury inflicted in the inocula- 85 tion process as reflected by a much lower germination rate.

SEED) IMOCULATIOMS. 1951 A. second inoculation process, Method B, was devised in May, 19515 for mass infection of seeds. It was considered desirable to inoculate comparatively large numbers of seeds simultaneously and to avoid repeated periods of surface sterilization after inoculation. The hypodermic inoculation method was tedious, complex, and limiting in the number of seeds that could be treated satisfactorily. The drilling apparatus in Figure 12 facilitated rapid preparation of seeds for inoculation. An electric hand-drill was clamped to a leg of a tripod and adjusted in place to have the drill-point protrude in a seed-shaped cavity in the upper side of the drill-board or platform. The seed, placed on the brass wire spring across the cavity, was pressed down against the drill-point which penetrated the seed to a depth of approximately two millimeters midway between the chalazal and micropylar ends of the seed. Spring tension removed the seed from the drill-point. Seeds were surface-sterilized in mercuric chloride before and after drilling; the drill-point and drill-board were frequently surface-sterilized with 70 percent ethyl alcohol.

Drilled seeds were placed in sterile, wide-mouthed bottles and sufficient inoculum, consisting of an aqueous suspension of conidia and mycelial fragments from two-weeks- 86 old colonies of Verticillinm (Tipton strain) grown on pota­ to-dextrose agar, was poured Into each, bottle to entirely Immerse the seeds. The number of conidia and mycelial fragments In each milliliter of Inoculum was determined by counting In an " Improved Neubauer hemocytometer with Levy Counting Cham­ ber, Three separate counts were made and averaged. This lot of Inoculum contained 1,810,000 conidia and mycelial fragments per ml. The bottle, containing the Inoculum and seed was plugged with a sterile cork and shaken well to remove air bubbles from micropylar and wound openings. To force In­ oculum Into these openings the air In the bottle was evacu­ ated by vacuum provided by an Airejector water-faucet pump. Evacuation was performed three times, twice for 5 minutes, and once for 10 minutes. Seeds and inoculum were strongly agitated by shaking between evacuations. After atmospheric pressure was restored In the bottle the excess Inoculum was poured off. Seeds were removed to a sterile dish contain­ ing dry, sterile, filter paper where the seeds were dried for 12 hours. Seeds were protected from dust by six layers of cheesecloth previously soaked In 1:1000 mercuric chloride and wrung practically dry. When dry, seeds were then sur­ face-sterilized a final time in mercuric chloride for 3 minutes, dried again, and stored In sterile containers loosely stoppered with cotton wool. 87 Tests were made of the efficiency of this inoculation method by culturing seed on agar and on sterile filter paper in Fetri dishes. Fifty of seventy seeds (71 percent) inocu" la ted by this process produced.' the fungus in 10-days time. All of the tested seeds ge rmihated, ■ Other inoculations for use during the planting season of 1951 were made as shown by Table 8. Seeds for planting as controls .were s1ml1arly^ t^ but vacuumed in sterile distilled' water' instead of fungus inoculum. ^ S':;;/

SEED INOCULATIONS. 1952' The methods of seed inoculation empldÿèd in 1950 and 1951 proved generally unsatisfactory. Wounds inflicted in the seeds created an unnatural condition, which evidently contributed to the early decay of the embryo by wound para« sites. Mechanical injury to the embryo by the pénétration of the hypodermic needle or the drill-point in the previous­ ly described inoculation methods showed its effects in re­ duced germination rates of both inoculated and unino cula ted seeds, particularly intihe long-staple cotton varieties. Two separate observations made during the first two inoculation processes suggested that a third method (Method C) might be employed to eliminate wounding of the seed. In soaking seeds to soften the seed coats in Method A, it was noted that the micropyles of the swollen seed opened and remained open until the seed dried. During vacuuming 88

TABLE 8

Drilled seed inoculated by Method B,

No. of Coni di al-myc elial Cotton seeds Fungus fragment count variety Date (Approx. ) isolate per ml.

Pima 32 6/20/51 350 Tipton 1,830,000 1^17 RB 6/20/51 Do Do 1 ,109,000 Pima 32 Do Do Gliocladium 1.420.000

1517 RB Do Do Do 1,420,000 Pima 32 6/21/51 Do Thomas 75,000^ 1517 RB Do Do Do 75,000^ Pima 32 7/11/51 Do CMI 1,275,000 1517 RB Do Do Do 1,275,000

Mycelium-fragment count since this isolate did not produce conidia. Mycelial mats in sterile distilled ■water were macerated and mixed by Waring Blender for 10 minutes prior to count. 89 employed to inoculate seeds by Method B it was observed that comparatively large quantities of inoculum were absorbed by the seeds. Simpson et al. (1^6) observed that seeds were not materially injured by soaking them in water and subject­ ing them to greatly reduced atmospheric pressure to facili­ tate water penetration of the seed. By combining these ob­ servations into a single process, a method was devised which produced inoculated seeds having approximately the same.appear­ ance as normal acid-delinted ones* The general procedure for inoculating the seeds without wounding them was identical to that described for Method B. Surface-sterilized, uninjured seeds were placed in inoculum where they were soaked for fifteen and one-half hours. After 2 hours in the inoculum, the air in the container (a wide­ mouthed bottle) and the seeds was partially evacuated. Evacuation was repeated at twelve hours and at fifteen hours. During each air evacuation the container was agitated period­ ically to assist in dislodging the bubbles of air formed at the chalazal and micropylar ends of the seeds. Replacement of the air originally in the seeds by inocul’um was indicated by the reduced amount of free liquid inoculum in the container. A preliminary test was conducted to determine the quanti­ ty of inoculum that would be required to treat a given number of seeds by the above method. Seeds were soaked in tap water for 2h hours, during which they were air-evacuated three times. 90 This test indicated that 100 ml. of inoculnm would be suf­ ficient to treat approximately 300 seeds. Two 100 ml. quantities of inoculum were prepared from macerated cultures of the CMI strain. By the hemocytometer method the number of conidia in suspension was determined to be approximately 30,000,000 per ml. Two hundred and., fifty seeds of each of the two cotton varieties, 1517 RB and Acala M+, were inoculated, dried, surface-sterilized in.mer­ curic chloride, rinsed in sterile distilled water, again dried, and stored in sterile bottles. Four days after inoculation, 128 seeds were immersed in mercuric chloride for 1 minute and cultured on potato-dextrose agar in Petri dishes. At the end of 12 days, the inoculated fungus had been recovered from 126 seeds. The two seeds not successfully inoculated were small and hard and showed no evidence of having opened at the micropylar end during soak­ ing. Each of the 126 successftilly inoculated seeds was dis­ sected and examined for evidence of fungus growth. Table 9 shows that the fungus frequently was obtained from more than one location. In 109 cases alone the fringe tissue was found to be invaded when examined microscopically. The results of this seed inoculation process indicated that the thin membraneous tissue, generally regarded as the remains of the nucellar sac, most frequently supported fungus growth. Attacks by the fungus on the cotyledons’ 91

mBLB: 9 Results of eulturirig seeds inoculâtedl by Method C.

Acala 44- Total Number p.f seeds cultured 48 80 128

W , 78 126 Germinated 120 Germination interrupted 1 Cotyledons_ attacked with séëd 'coët 'àttadhed 6 ' 11 17 Cotyledons attacked with seed, coat di-opped , . ,,,, 2 11

Fringe tissue invaded 68 109, .

Root Infections 3 16 Agar streaked with ftingus, , seedling not attacked ' 6 27 33 Agar streaked with fungus, seedling attacked 6 21 27 92 occurred only in those cases where the seed coat remained attached to these structures or those in which the cotyle­ don was injured in attempting to shed the seed coat (Fig. 1 3 )• In a number of cases, it was noted that the fungus was carried from the interior of the seed out onto the agar by the developing seedling, apparently without causing in­ fection to the seedling. In a few instances the fungus growth, originating in streaks on the agar, eventually attacked the seedling causing death (Pig. 1^). Only 8 seeds failed to germinate; however, germination was interrupted by fungus attack of the seedling in 9 cases. The occurrence of fungus growth in the cast-off seed coat was common, particu­ larly appearing at the chalazal end. Further evidences of resistance to infection were observed in the occurrence of gray-white, greasy-appearing masses of Verticillium growth in the folds of the cotyledons and at their juncture with the hypocotyl. Verticillium was observed on the agar direct­ ly in contact with the hypocotyl, but no lesions occurred and infection was not evident. Only in a few cases were the seedlings attacked by the fungus through the roots.

FIELD AND GREEKHOÜSE STUDIES GREENHOUSE ELANTIHTGS - INOCULATED SEEDS. 1950

In order to determine whether the fungus inoculated into seeds by Method A could produce diseased seedlings or mature plants, a number of these seeds were planted periodically in 12-inch pots of sterile soil in the greenhouse. 93 !Ehe soil used was a mixture of 3 parts heavy adobe soil, 2 parts sand and 1 part well-rotted manure. After mixing and placing soil in clay pots sterilization was accomplished by autoclaving at 2kh degrees P. for 8 hours. Seed of the varieties SxP, Pima 32, 1517 RB, and Paula C, inoculated with Tipton strain were planted at the rate of 15 seeds per pot. Surface-sterilization of the seed before planting consisted of immersing them in mercuric chloride (1:1000) for 30 seconds. Seeds were rinsed twice in sterile distilled water. Sterile forceps were used to handle seed during the planting process following sterili­ zation and rinsing. Seeds were placed on a lightly-tamped and firmed soil-surface in the pot, covered with one-half inch of sterile soil and one-quarter inch of sterile peat moss. Two pots of each inoculated variety of seed were planted as was one control pot for each variety consisting of 15 seeds hypodermically inoculated with sterile distilled water and otherwise handled exactly as fungus-inoculated seeds. Soil at the time of planting was sufficiently moist to preclude irrigation before emergence of the seedlings. After emergence plants were surface-irrigated with tap water as needed. Young seedlings were found bearing Verticillium at the wound-site on the cotyledons as shown in Tables 10, 11, 12 and 13. Growth was limited to the tissue immediately surrounding the wound and disappeared in 3-^ days. TABLE 10 ISEumbers of infected cotton plants in greenhouse resulting from Verticillium-inoculated seeds.

Indcu-^ - "Date" ''' No. of : "Nor of COtyle- lated inocu­ Date seeds emerg­ dpnary variety lated planted planted ences infections

- 1 - •' Q SxP 4 / 5 / 5 0 5/4/50 "^0 "

Control 5/1/50 Do 1 5 1 4 Ô Pima 32 4 / 1 2 / 5 0 Do 30 23 2 Control 5/1/50 Do 1 5 8 6 1517 EB 4 / 1 4 / 5 0 Do 30 27 2 Control 5/1/50; Do 1 5 1 5 0

Paula C 4/'2o / 5 o Do 30 19 5 Control ^ 1 / 5 0 Do 1 5 13 6

TOTALS’ 13^ 9 95

i!EA.BLE 11 Numbers of infected cotton plants in greenbonse resulting from #erticilllum4inoculated seeds*

ÉQOcur m t e No * o f No. of Cotyle- lated inocu­ Bate seeds emerg- donary ^ a r i e ^ lated planted : planted .ences infections

SxP V 5 / 5 0 ; 5/ 12/50 3(m. ; 19 0 Control 5/1/50 Do 1 5 6 B 0 32 lf/12/50 Do 30 22 0 Control 5/1/50 Do 1 5 13 0 1517 # V 1V 5C1 Do 30 22 1 V

Control 5/1/50 Do 1 5 Ilf 0 G V 2 0 / 5 C > Do 30 ; 18 1 Control 5/1/50 Do 1 5 T 12 j 0

.. ' . T01M.S; 180 1 # i 2 TABLE 12 Numbers}of infected cotton plants in greenhouse rresn^^ltih^ f"r Vertidill ium-i-nb cnlated seeds.

Inocu- Date No. of No. of Cotyle- lated inocu- Date seeds emerg­ ddnary variety lated planted planted r ences infections

6 / 1 6 / 5 0 " 30/" 13 Q Control ^ / 5 o Do 15 8 0) Pima 32 V 12/50 Do 30 15 0 Control # 1/50 Do L5 10 Qi 1517 EB V 1 V 9 0 Do 3 0 27 2 Control 5/ 1 / 5 0 Do. 15 1 4 0) Paula C 4/20/50 Do 30 23 3 Control 5/1/50 Do 15 13 0 t o t a l s ; I8à 123 5 97

•; TABLE 13 Numbers of> infected cpttomiplants in greenhouse resulting from Vertic'ililum-inoculated seeds;

Inocu­ ■ Date No, of No.-of;: Cotyle- lated inocu­ Date seeds emerg­ denary : variety lated planted planted ences infections

S x P V 5 / 5 0 7/ 8/ 50; 30 8 0 Control 5/Î/5Q) Do 1 5 11 0

Pima 32 V 12/50 Do 3 0 1 7 0 Control 5/1/50 Do 1-5 12 0

1517 RB 4./lV:50) Do 30 27 0 Control 5/1/50 Do 1 5 13 0 Paula C 4/20/gO) Do 30 2k- 0

Control 5 / 1 / 5 0 Do 1 5 I k 0

TOTALS 180 126 : 0) , " 98 The necrotic tissue fell out leaving a small shot—hole approximately one-eighth inch in diameter. Plants of these four series were allowed to grow until approximately 9 months of age when the experiment was dis­ continued. Earing the entire growing period no symptoms of Verticillium wilt were observed. Cultures made of a repre-

■ ■■ sentative number of plant stems of each series C25 total) produced no Verticillium. The fungus, Gliocladium roseum. was isolated from the lower stem pieces of 7 plants of this culture series. This fungus was mistakenly identified as Verticillium sp. because of the verticillate-like branching of conidiophores occurring when the fungus is grown under certain conditions in the laboratory. Following scrubbing of the stems in tap water, they were split longitudinally in search of vascular discoloration. Pieces one-half to one inch in length were cut from selected stems, surface:-sterilized in Badass disinfectant for one and one-half minutes after which pieces were rinsed in two changes of sterile distilled water. Pieces were transferred aseptically to Petri dishes containing potato-dextrose agar of pH 7.2 and cultured in an incubator at 23 degrees G. for

one month. It is evident from the results of this experiment that the method of inoculation did not materially lower the germination rate of the short staple varieties but caused considerable reduction of germination rate of the long- 99 srbapie seed. The rorigus: failed to cause disease in a single plant, even though the jplants were grown for a period of time much longer than the normal seasonal growing period of the host.

Cotton plants grown in the greenhouse during this: per­ iod were generally etiolated and chlorotic in appearance. Some initial stunting of plants grown fromi fungus-inoculated seeds was observed,hut soon these: plants were indistinguish­ able from control plants.

FIELD PLANTINGS - jŒOCÜLATED SEEDS. 1 9 % Field testing of seed inoculated by Method A was. con­ sidered necessary to determine whether the inoculant affect­ ed the host plant and transmitted wilt under field conditions. Additional lots of approximately UOO seeds of each variety, Pima 32 and 1 ^ 1 7 BB, were inoculated in the previously described manner. These varieties were selected in order to have one resistant variety (Pima 32) and one susceptible variety ( 1 ^ 1 7 RB), Analogous lots of seeds were prepared for planting as controls by drilling a small hole in the seedcoats of each seed with a No. 5^ wire drill mounted in a. Hamilton-Ross electrical handy-tool. The drilled hole in these seeds was comparable to those made by the hypodermic needle in the fungus-inoculated seeds in size and amount of injury to the embryo. Drilled control seeds were soaked in sterile distilled water for a period equivalent to that 100 req'utred for seed-coat softening in the inoculation process, A. field plot measuring approximately 30 by 220 feet on the University farm was secured. This plot had no Verticil- llum-susceptlble crops grown there for at least 14- years. The plot was tilled, laid out into 8 rows, and irrigated for planting. The diagram below shows the planting scheme. The ranaining half of the plot was a duplicate of the half- plot shown. XXX Eima 32 Cinoc.) xxx Pima 12 (Control) xxx XXX Pima 12 (Control)xxx Pima 12 (Untreated)xxx ^

XXX 15*17 RB (Inoc. ) xxx 1 ^ 1 7 RB (Control) xxx |

XXX 1517 RB (Control)XXX 1 5 1 7 RB (Untreated)xxx 4— -Irrigation flow The x*s at each row-end and middle indicate a 6-foot barrier or spacer of sorghum. Row sections planted to cotton were 50 hills in length; hills were 2 feet apart. Three seeds were placed in each hill and covered by hand. Planting occurred on July 7, 1950, and first emergences were observed 4- days later. Normal cultural practices were followed. The plot was observed regularly at weekly intervals for symptoms of infection; however, none was found. The number of seeds of each planted group, the number of emergences, and the per­ cent of emergence are shown in Table 14-. No unusual differences in growth characteristics were observed. The Pima 32 sections failed to mature in the 101

TABLE 1^

iÉiergences occurring in field test, 195®.

Variety Hfumber Emerged Percentage of planted number planted

inoculated seeds

Pima 32. 300 133 54.33^ 1 5 1 7 EB . 300 209 69*66 Control ;seeds Pima 32 600 316 52.66

1517 RB 600 ^29 71.50 Untreated seeds

Pima 32 300 179 59 • 66 1517 EB 300 236 78.66

^'Aipproximatèly 10 hilis washed out during irri­ gation/before final record of emergence was made. 102 short growing period allowed. Sections planted with un­ treated seed produced healthy plants. One dusting for aphid control was applied by hand duster; a mix of 10 percent DDT, 2 percent gamma isomer bensenehexachloride, and 50 percent sulfur satisfactorily controlled these insects. Examination of the plants for vascular discoloration was made at the end of the season (November 30, 1950). Plants grown from inoculated and drilled control seed were sliced longitudinally. Approximately 10 percent of the 1517 RB plants from inoculated seed showed an almost imper­ ceptible yellow-brown discoloration of the vascular system below and slightly above the soil line, SxP plants failed to show the discoloration as did those plants produced from drilled control seeds. Culture of a representative number of these plants failed to produce Verticillium; however numerous isolations of Fusarium were made.

FIELD PLANTINGS - INOCULATED SEEDS. 1951 Field planting of seeds inoculated by Method B was made June 28, 1951j except for seeds bearing CMI strain, which was obtained from England July 3, 1951. Transfers for reproduction of this isolate were made immediately and microscopic examination on J u l y 10 showed that sufficient conidia were present to warrant inoculation of seeds which was accomplished the next day. Ttiese seeds were planted in the field on July 12, 1951. A. field plot 300 feet long consisting of four borders of 8 rows each was prepared for cultivation by spreading two tons of cow manure on each border to stimulate and encourage vigorous and succulent growth. Borders were then plowed and disced. Kiree of the borders were further fertilized by the addition of ammonium sulfate at the rate of 400 pounds per acre. All borders were then harrowed and rows k-2 inches apart were laid out. The other border, having only manure added, was thus kept for comparison with the field planting of the previous year. Seeds, inoculated with the Tipton strain were planted in this border. Each border (divided crosswise into 2 sections of hills each) was planted according to the scheme shown below. The untreated section of Border 1 was planted with untreated SxP seed. Half of a border is shown; the remaining half was a duplicate of the first half.

XXX Pima. 32 (Inoc. ) XXX 1517 RB (Untreated) XXX

XXX Pima 32 (Control) XXX 1517 RB (Untreated) XXX N . XXX 1517 RB (Inoc.) XXX 1517 RB (Untreated) XXX

XXX 1517 RB (Control). XXX 1517 RB (Untreated) XXX <-— Water Flow Bbrder 1 was planted with seeds inoculated with the Tipton strain; Border 2: with the Thomas strain; Border 3 with Gliocladium. and Border 4- with the CMI isolate. Control rows were planted with drilled seed inoculated by vacuum with sterile distilled water. X's in the diagram represent 104- 6—foot row-sections of sorghum. Untreated 1517 BB section was planted, with healthy acid-delinted seed that were other­ wise untreated. Three seeds vrere planted in each hill by hand and covered.with one-half inch of moist soil. Due to hot dry winds occurring during this period it was necessary to irrigate the plot on July 1, only three days after planting, although the plot had been deeply irri­ gated 5 days previous to planting. Severe crusting of the soil materially reduced the emergence at the lower ends of rows (Inoculated-seed section)); raking of the seedbeds to break the crusts was ineffective. Inspection of the lower sections of the borders, planted with drilled and vacuum inoculated seeds, showed a very low emergence rate. Results are shown by Table 15* The upper sections of borders, planted with untreated seeds, had 87 percent emergence. Because of the low emergence rate in the inoculated sections, the lower 20-hill-half of each lower section was replanted on July 12, 1951, at the rate of 2 seeds per hill. Again-the emergence by hill count, including those seeds inoculated with CMI strain, was less than 25 percent. A number of seeds from both inoculated and control rows were recovered for laboratory examination 30 days after planting, from hills showing no emergence. A. majority of those seeds recovered were not germinated and all showed signs of gen­ eral decomposition. Cultures produced a number of different 105 - TABLE 15 Hills showing emergence ih days after planting seeds inocnlated by Method

Hiiis Planted Producing Percent Inoculated plants showing Variety with emergence

Border 1 ■

Pima 32" Tiptons . 8 0 . : 18 : ' 2 2 . 50 Do ' smfir 80) . 10 ■ 12 . 50 1 5 1 7 R B Tiptons 80 L' 19 ' 2 3 , 75 fJ'lDOi: y ' : SDW 80 25 _ . 31.25 TOTALS 320. 72 Average 2 2 . 5 0

k ' Border 2 Pima 32: Thomas 80 7 8.75

Do SDW; 80 13 16.25 1 5 1 7 R B Thomas 80 12 15.00 .::.,DP \ : SDW. 80 • 22 2 7 . 50 T p T # S 320 5^ Average 16.87 Border 3

Pima 32 Gliocladium 80 2.50

Do ■ s m 80 ■' - ' 17.50;

1517 R B Gliocladium 80 7 8*75 Do SDW 80 19 23.75 TOTALS 320 % 2 Average 1 3 . 1 2

^Border 4- not planted nntil the date this record was made, July 12, 1951.

Sterile Distilled Water 106 organisms probably contributing to decay, including species

of Rhizopus. Aspergillus ^ Pénicillium. Trichoderma and bacteria in profusion. Hematodes also had attacked the wounded seeds and were observed in test tube cultures. The few plants in the inoculated sections of borders were observed weekly until the end of thé season. No fol­ iar symptoms were produced during the growing season nor was definite vascular discoloration evident when plants were removed and sliced longitudinally in December, 1951* Cultures from 18 plants selected because of their stunted Condition produced negative results. Poor germination of inoculated seeds in the field made it necessary to test inoculated seeds on potato-dex­ trose agar to determine whether Verticillium or the wound inflicted during inoculation was the cause of the poor ger­ mination rate noted or whether other micro-organisms pres­ ent in the soil were responsible. Five each of Pima 32 seeds inoculated with CMI and Tipton strains and five each Of 1517 RB inoculated with CMI, Thomas, Tipton, and Glio­ cladium. were cultured on potato-dextrose agar slants fol­ lowing a two-minute surface-sterilization in Radars disin­ fectant. These seeds were not rinsed in sterile distilled water before being transferred by means of sterile forceps to slants. Test tubes were then placed in covered quart Mason jars containing two to two and one-half inches of 107 tap water and cultured at 23 degrees C. In 2^ days, Verti- cillium had grown from within 20 of 25 seeds inoculated and 21 seeds were germinated. Of the 5 seeds inoculated with Gliocladium all germinated and all produced Gliocladium in culture. Table l6 tabulates the results of this test. Growth of Verticillium from inoculated seeds occurred not only from the wound in the seed coat, but also from the micropylar and chalazal ends of the seed. Fungus growth was especially noticeable on the cast-off coats from the germinated seeds. Of particular interest was the fact that all strains of Verticillium grew readily in portions of the fringe tissue which remained attached to the cotyledons or in the inside of the seed coat. The effect upon the coty­ ledons was similar to that described in tests of hypoderm­ ically inoculated seeds in 1950. The fungus initially af­ fected the cotyledons only in the immediate vicinity of the drill-wound (Fig. 15). When the seedling was moribund as a result of sup­ pressed development, it was noted that the cotyledons and hypocotyl were completely invaded by the fungi; most rapid in action was Gliocladium which quickly reduced the seed­ ling to a brown watersoaked mass. This experiment conducted with small numbers of seeds, due to the fact that several groups of inoculated seeds of the lot were utilized in replantings in the field, showed that the method of inoculation was successful. This was TABLE 16 Results of culturès of Method B-inbcùlatéd seeds on potato-dextrose agar.

Product^ in oc, fungus Sëed variety Cultured ■ ■ Fungus Germ. Cdntâml- : bn and inoculum recovered nations Coty­ ; Fringe ' Seed ledon tissue coat

Pima 32;(CMI) 5 ' « :: 5 0 2 i 5 : 2 Do.(Tipton) S; 3 3 : 2 1 ' 3 1

Do (Control) % ; . 0 5 0 ; 0 0 1517 RB (CMI) 5 C 4 i ; 4 ' 1 0 : 1

Do (Thomas) 5 4 4 . 1 1 4 r: 2 Do (Tipton) 6, 4 - 5 0 1 4 2 Do (Gliocladium) 5 5 - 0 : 2 3

Do (Control) 5 0 ; 5: 0 : 0 0 :: 0

TOTALS 40 25 ^ 36 / 7 : 20 12 PERCENT GERMINATION: Pùhgus-lnocülatëd seeds; fB6.66Ipercent' Control (Water-inoculated seeds): 100 percent H § 109 Indicated by the b.1^ percentage of ’’takes”. Recovery of the fungus in pure culture in all except four instances showed that the inoculum penetrated the seed and that the pathogen, was able to exist inside the seed for approxi­ mately 3 months. The high percentage of germination indi­ cates that Verticillium and Gliocladium may be only indi­ rectly responsible for the poor germination rate experi­ enced in the field. This experiment also re-emphasizes the importance of the parts played by the seed coat and fringe tissue which support Verticillium and Gliocladium growth when other tissues do not. Of equal importance is the fact that the cotyledons and roots of young seedlings in test-tube cultures were not attacked initially. It may be reasoned therefore that plants might have been produced in greater numbers in the field had it not been for the pre-germination decay of the seed by soil micro-organisms other than Verticillium and Gliocladium.

GREENHOUSE PLANTINGS - IKOCULATED SEEDS. 1951. In order to compare germination and emergence results with those obtained in the field and in culture, seeds inoc­ ulated by Method B were planted in sterile soil in the green­ house (12-5-51). The soil, composed of three parts mesa loam, two. parts river-bottom sand, and one part well-rotted cow manure, in 12-inch clay pots, was steam-sterilized at 244 degrees P. for 8 hours. Fifteen seeds of each inoculated 110 group as sho-wn in Table 17 were planted in three pots

C5 seeds per pot):, following seed surface-sterilization in Rada's disinfectant for 2 minutes. Rinsing in sterile dis­ tilled water was omitted. ' First emergences were observed on the fourth day. From the fourth to the twelfth day small white fungus col­ onies appeared, forming a circle about the round wound made in the cotyledon by the drill (Fig. l6). 'White fungus rings frequently observed within one day disappeared by the following day. Scrapings made of all fungus rings present on the 12th day were examined microscopically. Many of the fungus growths proved to be Benicillium species; however, some of the colonies were Verticillium or Gliocladium. previously inoculated into the seeds. In cases where seedlings had not emerged, the seed planted at that site was removed and examined for viability and fungus effect. Several seeds were found to be "germin­ ated but had failed to emerge by reason of seed inversion in the soil; however, a number of seeds showed no sign of germination, and were decayed. Examination of germinated and ungerminated seeds (par­ ticularly those inoculated with the CMI strain of Verticil­ lium) showed fungus hyphae completely invading the embryo. After 21 days all emerged seedlings were removed for culturing. Seedlings were thoroughly washed in running tap TABLE 17

Results of plantIng seeds inoculated By Method B in sterile soil in the greenhouse, and of culturihg 21-day-old seedlings.

Seed varietyi No. seed Germl- Emer­ Cul­ Prod, inoc. fungus and inoculum planted nationsi*': gences tured By exam. By culture

Pima 32. (CMI) 15 8 6 6 • 1 2 Do (Tipton) 15 8 5 5 0 1

Do (Control) 15 10 6 6 0 0

1517 RB (Thomas) 15 15 14 14 0 2.

Do (Tipton) 15 11 9 9 0 1

Do (CMI) 15 10 6 6 3 2

Do (Gliocladium) 15 15 13 , 12 2 7

Do (Control) 15 13 11 11 0 0

Determined by removing seeds from location where no plant emerged, In« version of seeds reduced emergence rate.

H H H 112 water and Immersed In 1;1000 mercuric chloride for 4 min­ utes, rinsed In sterile distilled water, and cultured on potato-dextrose agar at 23 degrees C. Table 17 Itemizes the number of seeds germinating, the number emerging, the number cultured, those from which the inoculated fungus grew In culture, and the number of seedlings showing growth of Verticillium or Gliocladium on wounded cotyledons 12 days after planting. Wounding of seeds In the Inoculation process quite evidently reduced germination and emergence as shown by the results of planting control, water-inoculated seeds of the Pima 32 variety, Pima 32 seed had previously shown analo­ gous sensitivity to hypodermic Inoculation In 1950. It must be pointed out that recovery of the fungi In culture was made only from the cotyledons In the Immediate vicinity of the wound-site.

FIELD PLANTINGS - INOCULATED SEEDS. 1952 A third attempt to produce Infected plants under field conditions from Inoculated seeds was made In May, 1952. Seeds of two varieties, Acala 44 and 2916, were Inoculated April 30, by Method C. Approximately 1000 seeds of each variety were treated with each of the 5 Isolates, Thomas, Tipton, Mcîîeal, CMI and Gliocladium. Inocula were prepared from agar cultures as previously described except for the Thomas strain. In the latter case, 8 whole agar slants bearing dense mycelial growth and abun­ 113 dant pseudosclerotla were suspended in 350 ml, of sterile distilled water. The resulting suspension was transferred to a Waring Blendor glass jar which had been sterilized. The crude suspension was mixed three times for periods of tvjo minutes each in the Waring Blendor to produce a gray- black colored inoculum of creamy consistency. Microscopic examination of the resultant suspension showed numerous pseudosclerotia and mycelial fragments of sufficiently small size to enter the open micropyle of seeds immersed in the inoculum. Counts made by hemocytometer of the individ­ ual pseudosclerotia and hyphal fragments averaged 1,350,000 per ml, of inoculum, Conidia in suspension of inocula of other isolates were calculated as follows^ CMI, 17,500,000; MciTeal, 8,450,000; Tipton, 2,000,000; and Gliocladium, 13,650,000, Each 300 ml, quantity of inoculum contained the macerated colonies from 8 agar slants. Five thousand seeds were similarly treated in sterile distilled water for use as controls. Seeds were surface-sterilized after inoculation, dried, and stored in small paper sacks. Field planting was accomplished on May 7, 1952, in soil having no previous history of Verticillium-wilt infestation and on which only grain crops had been grown for 14 years. The field plot in which 4000 inoculated, and a like number of control seeds were planted, was laid out in four repli­ cates of ten rows. Each row was 120 feet in length. The 114 lower end of each rov/ was planted with 25 hills of 4 seeds each of one of the inoculated groups of seeds, A like num­ ber of hills of control seeds of the corresponding variety was planted at the upper end of each row nearest the irri­ gation- water source. A six-foot row-section of corn sepa­ rated the inoculated-seed hills from the o ontrol-seed hills. Ten days after planting the number of hills in which seedling plants appeared, was recorded. Of the 1000 hills planted with inoculated seed only 38 showed no emergence, or a hi11-emergence rate of 96,2 percent. In the control sec­ tion, 19 hills contained no plants and the hill-emergence rate was 98,1 percent. Of the 58 missing hills in the in­ oculât ed-seed section, 34 had been planted with Acala 44 variety. Those had been inoculated with the fungus strains as follows: Tipton, 4; McNeal, 7; CMI, 4; Thomas, 8j and Gliocladium, 11, In the rov;s planted with 2916 variety, 4 hills contained no plants. In this latter group, 2 hills had been planted with seeds inoculated with Gliocladium, 1 with Thomas strain and 1 with CMI strain. The field planting was observed weekly throu^out the growing season for symptoms of Verticillium wilt, but none was found, Normal cultural practices were followed. . During the early part of the growing season it was noted that the 2916 variety produced plants averaging several inches taller and more robust than those of the Acala 44 variety. By mid- August no differences in growth were observable and all 115 mat-uring plants appeared vigorous and healthy with the ex­ ception of several small spots in which the plants were attacked by Texas root-rot fungus. Early in November the number of plants maturing in each of the sections (inoculated and control) were counted as shown in Table 18. Each plant stalk produced from an inoculated seed was cut off near the ground line and each stem was examined Internally for wilt symptoms. The re­ sults of examination of 2590 plants were negative,

GREENHOUSE PLANTINGS - INOGTJLATED SEEDS. 1952 As a check against the results of the field planting, seeds of the same lots planted in the field were grown simultaneously in 120 twelve-inch pots of sterile soil in the greenhouse. Ten pots were planted May 15 with 5 seeds of each of the ten inoculated-seed groups and two control groups representing two cotton varieties and five fungus strains. The first emergences in the greenhouse planting were observed after three days. At 21 days each seedling was removed from the soil and examined for fungus growth and symptoms of Verticillium wilt. Negative results were obtained. The experiment was repeated July 21 in the same pots of soil which were first fumigated with Larvacide at the rate of one 2.5 ml, injection per 12-inch pot of soil. As in the first trial no Verticillium growth was found on the devel- TABLE 18

Results of field planting, 1952,

C ott on Strain of Hills No, of Hills showing Plants variety inoculum planted seeds planted emergence maturing

2916 Tipton 100 400 100 313 Do .McNeal Do Do Do 301 Do CMI Do Do 99 318 Do Thomas Do Do 99 314 Do Gliocladium Do Do 98 300 Do SDW* 500 2000 490 1476

Acala 44 Tipton 100 400 96 193 Do McKeal Do Do 93 244 Do cm Do Do 96 244 Do Thomas Do Do 92 220 Do Gliocladium Do Do 89 143 Do HDf* 500 2000 491 1223

%Control seeds inoculated with, sterile distilled water.

Percent plants produced by inoculated seeds: 2916— — — 77,30 Acala 44— 52,20

Percent plants produced by control seeds: 291673,30 Acala 44-- 61,15 H H ON 117 oping seedlings. The nnmber of emergences was recorded at iS days after planting in both trials and is listed by Table 19. All plants of the second trial were allowed to grow until mid-November when the plant stems were examined for internal discoloration. No typical symptoms of Verticillium infection were found. Reduced numbers of emergences occurred when seeds were planted 11 weeks after inoculation. Comparable reductions. also occurred in the controls. Only CMI-strain-inoculated seeds of the Acala kh variety appeared seriously affected, but the evidence obtained does not warrant attributing the results to the inoculum since no infections were observed either in the seedling or mature plant stages.

FIELD INOCnLATIONS. 1951 Beginning in September, various plant parts were hypodermically inoculated with aqueous-suspension inoculum, prepared as described previously. Counts made by heraocy- tometer averaged more than one million conidia and mycelial fragments per ml. of inoculum except in the case of Thomas strain. Mycelial-fragment counts for this inoculum averaged 7^,000 per ml. During a four-week period inoculations were made into plants produced by untreated seeds by the following methods; 200 bolls of different ages by penetration of the boll peri- 118

TABLE 19 Results of planting unwounded inoculated seeds in sterile soil in greenhouse.

Cotton Strain Emergences per 50 seeds at 18 days variety inoculum First trial Second trial

2916 Tipton 50 4-3 Do McReal 50 4-8

Do CMI ks 4-9 bo Thomas lf9 4-5 Do Gliocladium 4-7 4-4-

Do SDWa- 4-9 4-3

Acala Tipton 4-7 33 Do McNeal 44. 4-5 Do CMI. 4-6 23 Do Thomas 4-8 36

Do Gliocladium 4o 33 Do SDW^ 4-7 38

Seeds inoculated with sterile distilled water. 119 carp directly into a boll locnle; 297 boll pedicels, by pene­ tration of the pedicel midway between the boll receptacle and pedicel node; 100 fruiting branches, penetrations being made near the main stem-node; 100 main stems, within six inches of the ground line; and 100 main stems, within 8-10 inches of the stem tip. Inoculations were made of both SxP and 1517 RB varieties of cotton and represented 4- strains of Verticillium (Tipton, Thomas, Me Neal and C5MI) and one of Gliocladium in approximately equal numbers. Twenty control inoculations of each type were made with sterile distilled water for comparison. Field examinations of the two types of stem inoculations one month after in;) action disclosed that 75 percent of the lower stem inoculations and 67 percent of those in the upper stems were successful. Vascular discoloration was traced from the inoculation point on lower main stems upward to within 6 inches of the main stem-tip for all Verticillium isolates except Tipton strain. Discoloration of the wood by the latter^ was slight. The highest point discolored in a main stem by this, strain was approximately 10 inches above the inoculation point and more than 12 inches below the stem-tip (Fig. 17) • Discoloration caused by Gliocladium in these parts seldom exceeded 6 inches above the inoculation point, but commonly occurred for that distance in both directions from

the wound. 120 Verticillj-um strains affected the lower lateral bran­ ches of plants after inoculation was made on the lower part of the main stem. Discoloration of the vascular .system was found passing through branch traces into the branches. Cultures confirmed that the discoloration was caused by Verticillium.

Vascular symptoms resulting from upper stem-inocu- lations were generally restricted to the immediate vicin­ ity of the inoculation wound. Discolored vessels usually were found above or below the wound for a distance of 2 to 3 inches. In several instances, discoloration was noted extending 10 to 12 inches below the wound.

Cultures were made of 6 plants of each lot inoculated at the lower stem with Verticillium strains. The fungus was recovered from main-stem portions one inch long at various heights above the inoculation point to within 6 to 8 inches of the stem-tip except for Tipton and Thomas strains, both of which proved most difficult to reisolate in culture from pieces taken more than 6 to 8 inches above the point of injection. McNeal and CMI strains were recovered from stem-pieces showing no vascular discoloration. Cul­ tures of stem-pieces taken below lower stem inoculation points were frequently over-run by Fusarium colonies. Pro­ fuse infection of field-grovm plants by Alternaria species caused much difficulty in reisolation attempts. 121 Thirty inoculated, branches were similarly cultured in efforts to trace the path of the fungus to the bolls. Ten

each were inoculated with McITeal, CMI, and Gliocladium. Strain CMI was recovered in 8 of 10 attempts and Glio­ cladium was reisolated in 3 of 10 attempts. Efforts to reisolate MeNeal strain were defeated by rapidly growing Alternaria colonies in all 10 cases. Reisolations of Thomas and Tipton strains from branches were not attempted. Bolls were collected from the 30 inoculated branches, tagged, numbered, and placed in small paper sacks for ex­ amination and culturing. In each case, the boll nearest the branch inoculation-point was selected. Each boll was examined for moisture content, maturity, lint condition, discoloration of the receptacle and pedicel, and for signs of the fungus in the placental column. Most of the bolls dried moderately between picking and examination before culturing. No conclusive discolorations were found in the receptacles and pedicels of these bolls. Locks of lint were removed from each boll and placed in numbered sacks corresponding to a. number assigned the boll. Bolls were then dissected:; the bracts were removed by trimming with scissors, the calyx was peeled away, the receptacle broken away from the carpels if dry and cut apart if moist, and the pedicel was cut off immediately below the receptacle. Placentae were cut from septa. 122 leaving only a small part of the septum attached.. The placentae, receptacle, and pedicel of each boll were then surface-sterilized by soaking in Rada’s disinfectant for 2 minutes. Pieces were rinsed in sterile distilled water and plated on potato-dextrose agar, slightly acidified with 2 percent lactic acid. Seeds were prepared for culture by ginning the locks from each boll separately in a small power-driven cotton gin, delinting in concentrated sulfuric acid, washing In running tap water for 5 minutes and air-dried. Seeds were then stored in small manila envelopes until May, 1952, when seed culturing was begun. Potato-dextrose agar was made of potato infusion, 2 percent dextrose and 1.5 percent agar adjusted to pH 7.0. Each seed was cultured on an agar slant after surface-sterilization. To facilitate sterili­ zation of all seeds of a boll simultaneously, the wire of a small kitchen strainer was removed and replaced by sewing four thicknesses of cheesecloth to the rim (Pig. 18). Seeds were placed in the cheesecloth strainer for immersion in 1:1000 mercuric chloride for 2 minutes, during which time the seeds were agitated in the disinfectant by shaking. Rinsing of seeds was accomplished by removing the strainer bearing the seeds to a small bowl containing sterile distilled water where the seeds were again agitated, Flamed forceps were used for transferring the seeds indi­ 123 vidually to agar slants. Fresh quantities of mercuric chloride solution and sterile distilled-water rinse were used for each 5 lots of seeds. Sterilizing and culturing of seeds was performed in a culture chamber previously de­ contaminated by washing all surfaces with a 5 percent form­ alin solution. The slants were incubated at room tempera­ ture (approximately 2? degrees C.> for 28 days and were ex­ amined for germination and fungus growth at weekly intervals. Tables 20 and 21 compile the results of culturing inoculated branches, bolls from those branches, the description of the individual bolls, and culture results from boll parts and seed. Pedicel inoculations were observed periodically in the field. One week after inoculation it was noted that slight wilting had occurred for all Verticillium strains as^ well as fop? pedicels inoculated with Gliocladium. Wounds in all cases were generally callused; however, bracts had fallen from a number of McNeal-inoculated bolls, but were not par­ ticularly affected by the other strains of Verticillium or by Gliocladium. Droppage of bolls due to pedicel injury was negligible. Two weeks after pedicel inoculations were completed, ten bolls of each inoculated group were collected and taken to the laboratory for examination. Vascular discoloration of boil pedicels and receptacles was most noticeable in those having been inoculated with the CMI strain of Verticillium; TABLE 20

Culture of branches and branch-inoculated boUs*

P l a n t Description Verticillium isolates and boll numbers T o t a l s

o f

p a r t s b o l l p a r t s

and culture M c N e a l CMI

s t u d i e d r e s u l t s

1 2 3 4 5 6 7 Ô 9 1 0 21 22 23 24 25 26 27 28 29 30

B o l l M a t u r e X XX XX X X XXX 1 0

D o I m m a t u r e XXXX X X X X XX 1 0

D o D r y XXXX X X XX X 9

D o M o i s t X XX X XXXX X XX 1 1

L i n t C l u m p e d X XXXXXXXXX X X XX 1 4

D o D i s c o l o r e d XXXX XXX X X XX X X 1 3

B r a n c h C u l t u r e d XX X X X XX X X XXXXX X X X X X X 2 0

D o Fungus recovered XXX XXXX X 8

P e d i c e l C u l t u r e d X X X XXXXXXX X X X X X X X X X X 2 0

Do Fungus recovered XX XX 4

Receptacle Cultured X XX X XXX X X XXXXX X X X XX X 2 0

D o Fungus recovered X X XX 4

P l a c e n t a e C u l t u r e d XXXX XX XXXXXXXX X X X XX X 2 0

D o Fungus recovered X 1

S e e d s C u l t u r e d 4 0 3 3 2 4 35 22 42 39 42 39 4 1 3 9 3 7 3 9 35 35 33 36 37 39 38 721

2 0 Do Germinated 0 11 0 34 0 41 15 42 27 3 3 9 38 13 28 30 34 36 35 38 4 8 4

Do Fungus recovered 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 H r o - r TABLE 21

Culture of branches and branch-inoculated bolls.

P l a n t Description Fungus isolate and boll numbers T o t a l s

o f

p a r t s b o l l p a r t s

a n d Gliocladium

s t u d i e d c u l t u r e

r e s u l t s

11 12 13 14 15 16 17 18 19 20

B o l l M a t u r e XXX 3

D o I m m a t u r e XXXXX X X 7

D o D i y XX XXX 5

D o M o i s t X XX X X 5

Lint Clumped XX XX XXXX 8

Do Discolored X XXX X X X 7

Branch Cultured X X X X X X X XXX 1 0

D o Fungus recovered X XX 3

P e d i c e l C u l t u r e d X X XX XX XXXX 1 0

Do Fungus recovered X XX 3

R e c e p t a c l e C u l t u r e d XXX X X XX XX X 1 0

D o Fungus recovered X XX 3

P l a c e n t a e C u l t u r e d X X XXXX X X X X 1 0

Do Fungus recovered XX X 3

S e e d s C u l t u r e d . 3 6 4 0 3 2 3 0 39 39 34 39 24 39 3 5 2

Do Germinated 36 3 8 3 2 0 38 24 33 34 0 1 9 2 5 4

D o Fungus recovered 0 0 0 0 0 0 0 0 0 0 0 126 however, discoloration was present in those inoculated with the other Verticillium strains, though to a lesser degree, especially in the case of the Tipton strain. Gliocladium caused a distinct browning of the pedicel and receptacle (Fig. 1 9 ) and of the lower half of the placentae nearest the receptacle. The lower part of locks of Gliocladium. pedicel- inoculated bolls was yellowed. Gliocladium was found grow­ ing and producing conidia at the bases of, and along the infolded edges of, the placentae nearest the receptacle. Several partially opened immature bolls were found to bear the fungus in the pithy central core, particularly in the lower part near the receptacle. Seeds in discolored locks were attacked and killed. A- similar lot of bolls collected the third week after inoculation showed that internal discoloration of pedicels and receptacles was much more evident in all cases than had been found the previous week, CMI strain of Verticillium had caused yellow-brown discoloration of the lower half of locks. At first septa appeared oil-yellow to citrine; later chestnut-brown (l40). Placentae and funiculi were browned and seeds in the lower half of locks had ceased development. McNeal strain caused only slight discoloration of the in­ ternal tissue of the receptacle and of the lower parts of the placental column. Lint in the majority of cases appeared slightly yellowed at the lower tip of the lock in the region 127 of the pit.

The Thomas strain had caused a distinct brown dis­ coloration of the receptacle, and lint in the pit region of the boll locules was pale gull-gray in color, water- soaked, and of reduced strength. Those bolls bearing the Tipton strain from pedicel inoculations exhibited symptoms very similar to those caused by the Thomas strain. In November, twenty bolls of each of the pedicel- inoculated groups were collected at random, tagged, number­ ed, and placed in separate sacks as had been done with branch-inoculated bolls. Some of the bolls collected at this gathering were immature and moist, while others were definitely dried and opened fully. All were stored at room temperature in the laboratory for several days prior to examination and processing. It was found that the storage in manila-paper sacks constituted, in effect, placing the bolls in a moist chamber,since sufficient moisture was retained to favor growth of Verticillium and Gliocladium on various parts of the stored bolls. Verticillium strains were easily scraped from the placental column and pithy boll core and in several instances were found growing from the portion of the carpel tissue nearest the receptacle. Num­ erous locks of lint were found discolored grayish black (lower half) and on these Verticillium was found growing

(Fig. 20).’ and producing pseudosclerotia, except in the case 128 of CMI strain which was never found to produce these bodies,

(Figs. 21 and 22). Gliocladium also was found growing from all parts of several pedicel-inoculated bolls in dense pinkish white col­ onies. Growth of this fungus was most abundant and no tis­ sue appeared free from invasion. Locks of lint were partic­ ularly affected and supported heavy mycelial growth which soon appeared light congo-pink (l4o). Cotton fibers not visibly supporting growth of Gliocladium fungus were shrunken, yellowed and weakened. Fibers taken from the area supporting fungus growth were mounted on microscope slides in Sartory’s solution containing 0.25 percent Orseillin BB (Alcorn and Yeager, 1). Fungus hyphae were found inside the fiber CFig. 2 3 ) and in some cases appeared to completely fill the lumen of the fiber. Penetration of the fiber walls was noted and, in one case, the branching of conidiophores and conidial production were observed inside the lumen. The green carpels of immature bolls appeared readily attacked by Gliocladium and became water- soaked and dark-colored, from brown to almost black. After observing that the path taken by the fungus from the inoculation point on the pedicel, through the receptacle and into the locule of the boll, appeared to involve passage through the pit at the base of each locule, it was decided to check the extent and path of penetration. This was 129 accomplished by the use of a water-soluble dye.

Forty bolls of different stages of maturity taken from 1517 RB variety of cotton were collected from the field and taken to the laboratory for inoculation studies. An aqueous solution of 0.1 percent gentian-violet. stain was prepared and inoculated into boll pedicels. The amount of stain in­ jected corresponded in quantity to that normally injected when using inoculum in the field (approximately 0,1-0,2 ml,). Pedicels were inoculated in groups of 5 and bolls were immediately sliced longitudinally to determine the path of the stain in the boll. Of these, 37 showed no staining of the lint at the base of the locule. Two bolls which were somewhat shriveled at time of collection showed slight dis­ coloration of the lint at the locule base.

One boll pedicel was injected with 0.6 ml, of stain. This boll also showed staining of lint; however, the locks were not stained as much as expected. Only the lower one- fourth of the locks showed discoloration, whereas in Verticillium-inoculated bolls as much as one-half of the locks showed fungus growth and discoloration, including the production of pseudosclerotia. Other bolls collected in the field were injected with stain through the pedicel, after the boll had been opened and the fiber locks removed. When quantities of stain com­ parable to normal injections of inoculum were used, stain was observed to penetrate the thin membranous inner layer of 130 the pericarp which, separates the locale cavity from the dietyostele-type, suh-carpellary complex of the receptacle. Injection of stain through carpels of the boll direct­ ly into the locule revealed that the moist lint was only slightly penetrated by the stain. Most of the stain passed along the space between the lock and septum for almost the entire length of the boll* Stain was hot observed to pass through the septa to cause discoloration in adjacent locules Bye insertions into portions of stems showed that the strains spread through the vascular tissue for comparatively great distances from the injection point. In dry, woody stems, discoloration was measured 14- mm. below and 15 mm. above the inoculation point. Injections of stain through pedicels generally showed the coloration to be confined to the sub-carpellary com­ plex of the receptacle and to the carpel tissue, in which the stain appeared to follow vascular strands. Inoculation of bolls in which the inoculum was injec­ ted through the sides and tip resulted in premature opening and droppage, as well as some stunting. The greatest number of prematurely-opened bolls were those inoculated with Gliocladium. This fungus appeared quite capable of attacking all parts of immature bolls and was found pro­ ducing heavy mycelial colonies on bolls in the field. Verticillium strains did not produce comparable growth on bolls in the field. Gliocladium was especially evident 131 along the first snture to dehisce. New fungus growth appeared white at first, later congo-pink C l ^ ) , and sup­ ported numerous conidia. Not only was the locule into which the inoculum had been injected invaded but also adja­ cent locules were affected. Penetrations of the septa by the fungus was evident, and in one case, all five locules of the boll were attacked although only one locule had been directly inoculated. White fruiting growth of the fungus was found on the septa and in the placental column of this and other bolls (Fig. 2^), It was noted that fruiting growth of Gliocladium occurred most predominantly in the prematurely-opened, greener bolls. As might be e^qpected, lint was more severe­ ly affected than in bolls inoculated through the pedicel. However, the effect was similar and involved larger areas. Fibers were yellowed, water-soaked, clumped and decayed. Seeds in yellowed locks appeared to support growth of Glio­ cladium^ but upon closer examination it was noted that in most cases the seed coats were attacked. A few immature seeds from smaller, and evidently younger, bolls were com­ pletely invaded by the fungus. little fungus growth occurred in the field in Verticillium-inoculated bolls. When such material was placed in sacks and stored at room temperatures for several days, fungus growth (visible to the naked eye) appeared, but never in such quantities as in Gliocladium- inoculated bolls under similar conditions. 132 In the field, penetration of septa by Verticillium strains was shown by the discolored locules adjacent to those which had been inoculated. Discoloration was; most evident in the case of the Tipton isolate and least evi­ dent for the Thomas strain. Growth was supported in moist bolls and continued until the bolls dehisced, at which time growth in the lint abruptly ceased, as evidenced, by distinct demarkation of lock discoloration. Placentae were observed to be discolored! and supporting exceedingly fine, hyaline, arachnoid Verticillium hyphae. Bolls which were nearly mature at the time of inoculation showed only slight discoloration of the fiber locks and placentae.

m general, the effect of penetrating the carpel in the inoculation process showed that Verticillium is capable of penetrating septa, that moist immature lint serves as a suitable medium for the fungus, and that placentae may be attacked and support the fungus. Seeds from Verticillium-infected locks appeared to be litte affected, if at all; however, great variations in stages of seed maturity were noted. Examination of shrunken locks affected by Gliocladium showed that dis­ coloration and rotting of the lint occurred far more around the seed itself than was indicated by the external discoloration of the entire lock. Lint in the immediate vicinity appeared especially weakened, due to penetration of the fungus hyphae. 133 The 20 bolls of each pedicel-inoculated group, totaling 100 bolls were prepared and cultured as described for bolls and seeds from branch inoculations. Tables 22, 2 3 , 2*+, 25, and 26 show the results obtained. Boll descriptions listed by these tables were recorded at the time of collection in the field; lint descriptions were prepared when bolls were examined in the laboratory several days after collection. Fungus recovery from lint was recorded by microscopic examination of fibers.

All told, 2,967 seeds from bolls, pedicel-inoculated with Verticillium strains were cultured; 2,^24 (8^,0? per­ cent); of these seeds germinated, while Verticillium grew from 23 seeds (0.77 percent). From bolls inoculated with Gliocladium at the boll pedicel 739 seeds were cultured, 448 (60.62 percent)germinated, and 1?5 (20*97 percent) yielded the fungus in culture.

In all bolls inoculated either through branches or ped­ icels 4-,803 seeds were cultured, of which 3»507 germinated, 23 yielded Verticillium, and Gliocladium grew from 1^^. Verticillium-infected seeds germinating numbered 11, while 18 infected with Gliocladium germinated. IVhen seeds from immature bolls were cultured the number of germinations was substantially reduced, not as a result of infection by the fungi, but by reason of the immaturity of the seeds themselves. It should be noted that the CMI strain of Verticillium TABLE 22

Culture of bolls and seeds inoculated with Gliocladium through boll pedicels. •

P l a n t Description Boll nund?ers T o t a l s

o f

p a r t s b o l l p a r t s

a n d

s t u d i e d c u l t u r e 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

r e s u l t s

B o l l M a t u r e XXX XX XX 7

D o I m m a t u r e XXXXX X X X X XX X X 1 3

D o D i y X X XX XXX X X X 1 0

D o M o i s t X X X X XX XX XX 1 0

l i n t C l u m p e d X X XXX X XX XXX X X XX XX 1 7

D o D i s c o l o r e d X XXX X X XX X XX X X 1 3

L i n t Fungus recovered X XX 3

Pedicel Cultured XXX XX X XXX XX XXXXX X X X X 2 0

D o Fungus recovered X XXX X XXXX X XXXXXX X XX 1 9

Receptacle Cultured X XXX XXXXXXX XX XX X XX XX 2 0

D o Fungus recovered X XX X XX XXX X X X X X X 1 5

P l a c e n t a e C u l t u r e d X XXX XX X X X X XXXXX XXX XX 2 0

D o Fungus recovered XXX X XX XXXXX X X XX X XXX 1 9

S e e d Cultured 43 35 38 37 41 38 39 34 40 39 38 33 30 34 31 42 33 34 37 43 739

D o Germinated 39 34 33 36 40 8 7 31 36 3 2 0 6 8 14 35 31 26 34 25 448

D o Fungus recovered 0 0 0 4 0 19 7 3 0 23 29 27 15 18 8 0 0 2 0 0 155

H U) -T TABLE 23

Culture of, bolls and seeds inoculated with Thomas strain of Yorticillium through boll pedicels*

P l a n t Description Boll numbers T o t a l s

o f .

p a r t s b o l l p a r t s

a n d

s t u d i e d c u l t u r é 51,52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70

r e s u l t s

B o l l M a t u r e X XXXXXXX XXX 11

D o I m m a t u r e XXXXX XX XX 9 D o D i y X X X X X X XX X X 10

D o M o i s t X X XXX X XX X X 10

L i n t C l u m p e d X XX X XX XX XXXX X XXXX 17 D o D i s c o l o r e d XXX X XX XX X XXX XX 14

Do Fungus recovered 0

Pedicel , Cultured X XXXXXXX XX XX X XXXXX X X 20

D o Fungus recovered X X X XXX X 7

R e c e p t a c l e C u l t u r e d XXXXX X X X X X X XXX XXXX X X 20

Do Fungus recovered X X XX X 5

P l a c e n t a e C u l t u r e d X X X X X X XXXX XX X XXX XXXX 20

D o Fungus recovered X X X X XX 6

Sèed Cultured 35 38 44 30 4040 32 45 41 32 34 46 32 31 42 39 42 43 4132 759

Do Germinated 33 37 38 28 20 39 31 44 32 0 33 46 25 27 39 34 39 40 30 28 643

D o Fungus recovered 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 H w TABIE 2U

Culture of bolls and seeds inoculated vrith McNeal strain of Verticillium through boll pedicels

P l a n t Description Boll numbers T o t a l s

o f p a r t s b o l l p a r t s

a n d

s t u d i e d c u l t u r e 71 72 73 74 75 ?6 77 78 79 80 81 82 83 84 85 86 87 88 89 90

r e s u l t s

B o l l M a t u r e XXXXXXX XX 9

D o L m a t u r e X X XXX XX XX XX 1 1

D o D i y XXX X XX XXX X 1 0

D o M o i s t X X X XXXX X XX 1 0

Lint Clumped X X X XX X X XX X X X XX XXXX 1 8

D o D i s c o l o r e d X XXXX X XXX XXX XX X XX X 1 8

D o Fungus recovered X X XXX X X X 8

P e d i c e l C u l t u r e d XX X X X XXX XX XXX X XX X XXX 2 0

D o Fungus recovered 0

Receptacle Cultured X XX XX X XX X XX XXX X X XXXX 2 0

Do Fungus recovered 0

Placentae Cultured X X XX XX X X X XXXX XX X XX XX 2 0

D o Fungus recovered X XX X X XXXXX 1 0

Seed Cultured 35 36 39 23 39 41 41 2 9 3 9 39 39 37 29 3 6 3 3 3 6 3 8 3 9 3 0 4 2 720

D o G e r m i n a t e d 3 4 36 30 20 39 37 35 16 33 30 32 36 2 33 26 33 35 24 17 42 5 9 0

2 2 Do Fungus recovered 0 0 0 0 0 0 0 0 0 0 5 0 0 3 0 0 1 0 1 3

H (jj o\ TABLE 25

Culture of bolls and seeds inoculated with Tipton strain of Verticillium through boll pedicels

P l a n t Description Boll numbers

o f

p a r t s b o l l p a r t s

a n d

s t u d i e d c u l t u r e 9 1 9 2 9 3 9 4 9 5 9 6 9 7 9 8 9 9 1 0 0

r e s u l t s

B o l l M a t u r e X X X XXX XXX X

D o I m m a t u r e

D o D r y - X XX X X X X XX X

D o M o i s t

lint Clumped XXXX X X XX X X

D o D i s c o l o r e d XXX XXX X

D o Fungus recovered

Pedicel Cultured XX X XX XXXX X

Do Fungus recovered X

R e c e p t a c l e C u l t u r e d X XX X XXXX X X

Do Fungus recovered

P l a c e n t a e C u l t u r e d X X XX X XX XX .X

D o Fungus recovered X XXX XX

3 8 S e e d C u l t u r e d 4 3 4 0 3 6 4 0 4 2 3 9 4 5 3 9 2 9

3 8 D o GrexmLnated 4 3 3 6 3 6 3 9 3 6 3 8 3 7 3 3 2 5

0 D o Fungus recovered 0 0 0 0 0 0 1 3 3 /

TABLE 25 (cont.)

Culture of bolls and seeds inoculated with Tipton strain of

Verticillium through boll pedicels

P l a n t Description Boll numbers T o t a l s

o f

p a r t s b o l l p a r t s

a n d

s t u d i e d c u l t u r e 101 102 103 104 105 106 10? 108 109 110

r e s u l t s

B o l l M a t u r e X X 12

D o I m m a t u r e XXX X XX XX 8

D o D i y 10

D o M o i s t X XX X XX X X X X 10

L i n t C l u n q j e d X X X XXXX XX X 20

D o D i s c o l o r e d X X X XX XXX X 16 D o Fungus recovered X XX 3 P e d i c e l C u l t u r e d X XXXXX X X X X 20

D o Fungus recovered 1

R e c e p t a c l e C u l t u r e d X XXXXX X X X X 20

Do Fungus recovered 0

P l a c e n t a e C u l t u r e d XXX X X XX XX X 20

D o Fungus recovered XXX XXX 12

S e e d C u l t u r e d 39 38 35 34 35 40 35 34 34 35 759 D o G e r m i n a t e d 37 33 32 32 28 37 27 41 34 27 689 D o Fungus recovered 1 0 0 0 0 0 1 0 0 0 9 H % TABLE 26

Culture of bolls and seeds inoculated “with CMI strain of Verticillium through boll pedicels. .

P l a n t Description Boll numbers

o f

p a r t s b o l l p a r t s

a n d

s t u d i e d c u l t u r e n i 112 113 114 115 116 117 118 119 120

r e s u l t s

B o l l M a t u r e XXXXXX X XX X

D o I m m a t u r e

D o D i y X X XXXXXX X X

D o M o i s t

l i n t C l u m p e d XX XXX X XXX X

Do Discolored X X X XXX

Do Fungus recovered X XXXX

Pedicel Cultured XXXXXX X X XX

D o Fungus recovered

Receptacle Cultured XXXXXX X XX X

D o Fungus recovered

P l a c e n t a e C u l t u r e d X X XXXXXX XX

D o Fungus recovered X XXX X

Seed Cultured 36 34 38 37 36 32 27 40 44 31 D o C e z m n a t e d 36 32 36 37 35 30 25 38 42 29

D o Fungus recovered 0 0 0 0 0 0 0 0 0 0 H W v O TABLE 26 (cont.)

Culture of bolls and seeds inoculated vrith CMI strain of Verticillium through boll pedicels.

P l a n t Description Boll numbers T o t a l s

o f

p a r t s b o l l p a r t s

a n d

s t u d i e d c u l t u r e 121 122 123 124 125 126 127 128 129 130

r e s u l t s

B o l l M a t u r e 10

D o I m m a t u r e X XXXXXX X X X 10

D o D i y 10

D o M o i s t X XXXXXXX X X 10

Lint Clumped XXXX X X 16

Do Discolored X XXX X 11

Do Fungus recovered X XXXX 10

Pedicel Cultured X XXXXXXXX X 20

D o Fungus recovered 0

Receptacle Cultured XX X XX XXX XX 20

Do Fungus recovered 0

Placentae Cultured XXXXXXX X XX 20

Do Fungus recovered XXX X X 10

Seed Cultured 2 9 3 8 4 0 3 9 4 9 3 8 3 8 4 0 3 1 3 2 7 2 9

D o G e r m i n a t e d 1 9 16 8 22 2 4 3 1 3 5 4 0 22 2 7 602

Do Fungus recovered 0 0 0 0 0 0 0 0 0 0 0

H

g 141 was not recovered from a single seed. K"o explanation is offered for this phenomenon. Of some significance is the fact that bolls classed as "moist" were the majority of those from which the fungi were recovered in cultures of boll parts as well as of seeds. In contrast, 7 of the 9 seeds of the Tipton group from which the fungus was reisolated were obtained from bolls that were classed as "mature" and "dry". The Verticillium strains were observed producing colo** nies from one or more definite positions on the cultured seeds. Table 27 records the ihdlvidual boll numbers, the seeds from those bolls, the sites of growth of the fungus colonies, the infected seeds germinating, the fungus strain with which the boll pedicels were originally inoculated, and the presence or absence of the fungus in the boll prior to culturing the seeds. It is pointed out that only one seed originated from a boll in which no fungus was observed before the seeds were cultured, namely boll No. 107, inoculated with the Tipton strain,

GREENHOtJSE INOCXJIA TIONS. 1951 Inoculations of plant parts were made in the greenhouse where conditions of environment were controllable to some degree. This portion of the experiment had the added ad­ vantage of affording closer observation of individual inoc­ ulations. TABLE 27

Location of Verticlllium colonies growing from infected seeds.

Boll See^ Growth occurring at Germi­ Inoculum Verticillium observed . no* no. nated strain on lint or placentae before culture of Chalaza Micropyle Seed coat seeds in general

63 1 X 0 0 0 Thomas X

81 1 0 X 0 0 McNeal X

2 0 X X X Do X

3 X 0 0 0 Do X

4 0 X 0 0 Do X

5 0 0 X X Do X

84 1 0 XX 0 Do X

2 0 X 0 0 Do X

3 X 0 X X Do X

85 1 X XX X ■ Do X

2 0 X 0 0 Do X

0 Do X S 88 1 0 X X r o 2 0 X 0 0 Do X TABLE 27 (cont*)

Location of Verticillium colonies growing from infected seeds.

Boll Seed Growth occurring at Germi­ Inoculum Verticillium observed ' no. no. nated strain on lint or placentae Chalaza Micropyle Seed coat before culture of in general seeds

89 1 0 X 0 X McNeal X

97 1 X 0 0 X Tipton X

98 1 X X 0 X Do X

2 X 0 0 0 Do X

3 0 X 0 0 . Do X

100 1 X 0 0 X Do X

2 X X 0 X Do X

3^ 0 0 0 0 Do X

101 1 0 X X X Do X

107 1 X X 0 X Do Ô

TOTAL 23 10 15 11 22 H S

^Verticillium growth on agar; not observed elsewhere on seed or seedling. One hundred cotton plants (90 days old) of the 1517 RB variety were hypodermically inoculated with aqueous suspen­ sions of the fungi as had been done in the field. Twenty plants were treated with each of the four isolates, CMI, McNeal, Tipton, and G-liocladium. Inoculations were made of each of the following groups ; 5 bolls receiving injec­ tions througja the boll pedicel, 5 bolls inoculated through the pericarp directly into the locule, 5 main stems within 6 inches of the ground line, and 5. main stems 4 inches below the stera-tip* All inocula contained more than one million conidia and mycelial fragments per milliliter of suspension. Five inoculations of each category listed above were made with sterile distilled water for controls. The effects of the inoculations upon the stems and the lint were analogous to those occurring under field conditions. Bolls inoculated through the carpels and through the pedicels prematurely opened in a great number of instances and abscission of treated bolls was not uncommon, even with those inoculated with sterile distilled water. Gliocladium appeared to exert greater influence upon the bolls of both categories of inocula­ tions by causing the majority of bolls treated with this fungus to open prematurely and by producing the greater number of visible fungus growths on and in the affected bolls. Of the Verticillium strains, Tipton caused 100 percent premature opening of bolls in the pedicel- 145 inoculated series, while McNeal strain caused premature opening of 4 of the 5 bills inoculated through the boll carpels and produced visible fungus growth on these but failed to affect the fifth boll. Table 28 shows the number of premature openings, the bolls abscissed, a.nd the number of bolls upon which visible fungus colonies were observed. Pedicel-inoculât ions, , th o u ^ few in number, served to confirm the observation that penetration of the affected bolls (inoculâtions in the pedicel) apparently is effected èither through the small, pits in the portion of the locule immediately adjacent to the receptacle, or through the pithy center column of the boll. Both structures appear to be equally affected as indicated by discolora­ tion of these parts which has been observed in green, unopened bolls and in bolls that have opened at maturity.

PATHOGENICITY TEST BY HYPODERMIC IN0GX3IATI0N In order to re-check the relative pathogenicity of the fungus isolates under study, 15 vigorous plants of the 1517 RB variety of cotton growing in the greenhouse were hypodermically inoculated with aqueous suspensions of conidia and mycelial fragments. The plants had just begun to form squares at the time of inoculation. Inocula were prepared for three strains of Verticillium and for Gliocladium by macerating in 100 ml, of sterile distilled water 8 twenty-day-old colonies grown on potato- 146

T A m B 28 Resuits of boll and pedicel inoculations of greenbouse plants*

Inoculum Bolls Opened pre« Abscissed Colonies inocu- maturely visible la“ted in boll

Bolls inoculated through carpels

CMI 5 1 1 3 McNeal 5 4 0 4 Tipton 5 2 1 2 Gliocladium 5 4 1 4 SDW^ 5 1 2 0 Bolls inoculated through pedicels CMI 5 4 0 2 McNeal 5 1 2 1 Tipton 5 5 0 2 Gliocladium 5 4 1 3 SDW® 5 0 0 0

^Sterile distilled water 147 dextrose, agar slants. The Thomas strain formed no conidia and was grown in sterile tap water in a 250 ml. Ehrlenmeyer flask for 21 days to produce a dense mycelium of very fine hyphae which was shredded to form a suspension. A Bri^t-Line Improved Neubauer hemocytometer was used to determine the number of conidia per ml, of suspension. The following counts per ml. were recorded; CMI, 18,450,000; McIiFeal, 4,310,000; Tipton, 850,000; Gliocladium. 5,820,000. Thomas-strain suspension was not counted; however, sufficient growth was present in the 100 ml. quantity of tap water to cause it to have a milky-white appearance. Young, succulent stems and branches were injected with 0.2 to 0,3 ml. of the suspensions according to a method described by Evans (48) which was slightly modified; the part to be inoculated was first canpletely pierced with a sterile dissecting needle; the inoculum was injected through the wound made by the needle, and any excess inoculum flow­ ing through the wound was caught in cotton wool soaked in 70 percent ethyl alcohol. Three plants, each inoculated in 3 places, were injected with each of the 5 inocula. Three other plants, similarly treated with sterile distilled water, were established as controls. Two plants were not inoculated, but were wounded three times each with the dissecting needle as additional controls. The site of each wound was identi­ fied by a small tag. In ten days, wilting was apparent in all plants inoculated with.. CMI, McNeal, and Tipton strains . (Pig. 25%. Typical foliar discoloration occurred first in those plants receiving the Tipton strain. One plant injected with Thomas strain wilted at 15 days, one wilted at 15 days, and the third plant inoculated with this strain did not become diseased. Control plants into which sterile water was injected were not affected,.nor were those plants inoculated with G-liocladium roseum (Pig. 26) . Plants which were wounded with a dissecting needle only, callused promptly and showed no ill effects from the treatment. Pieces of leaf petioles collected 2 to 8 inches above the inoculation point from each of the inoculated plants were cultured on potato-dextrose agar. The pieces,.. approximately 1 inch long, were surface-sterilized in Rada’s disinfectant for 3 minutes and rinsed in sterile distilled water. Each portion of petiole was then asepti- cally cut into three parts of equal length. The middle part only was placed on agar and cultured at 23 deg. C. The Verticillium strains were recovered from each of the diseased plants. No fungi were recovered from pieces of petioles inoculated with Gliocladium or sterile water. All reisolated strains were similar morphologically to the parent culture except Thomas strain which now produced conidiophores and conidia in great numbers. In subsequent transfers of this strain on artificial media the ability to produce conidia was retained. The diseased plants in the greenhouse, growing in 12- inch clay pots, showed slight recovery from the disease 28 days after inoculation. At six weeks foliar symptoms had disappeared, although all infected plants were sli^tly stunted in comparison to control plants. To test the effect of nitrogen application upon the expression of symptoms, 4 grams of ammonium sulfate was added to each pot of soil, which was then surface- irrigated until excess water flowed from the drainage hole in the bottom of the pot. Foliar symptoms were again evident in 2 weeks and were most easily distinguishable on the plants infected with Tipton strain. Ko visible differences in pathogenicity were observed other than earlier and more clearly defined symptomatic expression occurring in plants inoculated jwith Tipton strain and the late appearance of symptoms resulting in the case of plants inoculated with Thomas strain. In addition, numbers of squares and small bolls were dropped. Approxi­ mately 30 of these, surface-sterilized and cultured on agar, did not yield Verticillium..

CULTURE OF YOUNG BOhIÜ FROM VERTICILLIUM-WILTBD PLANTS The isolation of Verticillium from young bolls was attempted for the following reasons; (a) numbers of squares and small bolls were dropped by diseased plants, and (b) 150 it was observed that immat-ure and moist bolls most readily supported growth of Verticillium following inoculations made in the field. Over an 11-week period, 410 young bolls were collected from greenhouse-grown, Verticillium-wilted plants. Bolls were collected as soon as the flower had discolored and wilted or fallen away. Each boll was snipped from the plant with scissors and immersed in tap water to prevent wilting. In the laboratory each boll was surface-sterilized in Rada’s disinfectant for 3 minutes and rinsed in sterile distilled water. Thereafter, bolls were aseptically removed to a sterile Petri dish containing four sheets of Whatman filter paper saturated with 1:1000 mercuric chloride, and each was sliced longitudinally through the boll and boll pedicel with a sterile scalpel. One-half of the boll was cultured on potato-dextrose agar by placing the freshly cut side in contact with the medium. The remaining half was killed in formalin-aceto-alcohol for future sectioning if the corres­ ponding half-boll produced Verticillium. Each cultured boll- half was incubated in a moist chamber at room temperature for 28 days. Examinations for Verticillium were made at weekly intervals. Of the 410 bolls collected and cultured, not one produced Verticillium from the boll portion. One isolation was made of Verticillium from a boll pedicel taken from a Thomas-strain-inoculated plant. PATHOGENICITY STUDIES OP ISOLATES USING- TOMATO. OKEA. A.NP E G G P L a M : ' :

Path-ogenicity experiments -were conducted with, tomato, okra, and eggplant, all of which are considered as being highly susceptible to Verticillium. The hosts included Earliana tomato. White Velvet okra, and Black Beauty egg­ plant. Twenty 8-inch clay pots of steam-sterilized soil were planted at the rate of 5 seeds per pot for each of the three hosts. Seeds were surface-sterilized in 1:1000 mercuric chloride for 1 minute and thoroughly rinsed in sterile distilled water. When plants were 8 weeks old they were hypodermically inoculated on the main stem 4 inches above the soil line. The inoculation method and preparation of inoculum are described in the section, "Pathogenicity Test- by Hypodermic Inoculation.", The concentrations of conidia and mycelial fragments were de­ termined in a manner already described. The counts for 5 inocula were; CMI, 50,000,000 per ml,; McNeal, 16,250,000 per ml.; Tipton, 4,500,000 per ml,; Thomas, 1,000,000 per mi.; and Gliocladium, 13,750,000 per ml. Approximately 0.1 ml. was injected into each host stem. In 21 days 90 percent of the inoculated eggplants showed typical symptoms of wilt caused by CMI, McNeal, and Tipton strains. Eleven percent of those plants Inoculated with Thomas strain were wilted, while those inoculated with Gliocladium and sterile distilled water showed no effect at the end of the same period. Tomato plants failed to show

foliar symptoms. Sli^t discoloration of the pith and vascu­ lar system was observed in the inmediate vicinity of the inoculation wound. One hundred percent of the okra plants inoculated with CMI, McNeal, and Tipton strains incurred the disease. Negative results were obtained for Thomas strain, G-liocladium, and sterile distilled water.

A FREVIOTTSLY UNDESGRIBED METHOD OF ISOLATING VBRTICILhlUM Because of the slow development of Verticillium in cultures prepared by' the previously described methods and those already published in the literature, a method per­ mitting easier and more fapid Isolation of the fungus from infected cotton stalks was sought. In August, 1952, thir­ teen plants showing foliar symptoms and internal discolora­ tion ascribable to Verticillium wilt were collected from the Sahuarita area, 25 miles south of Tucson, Arizona. En­ tire plants were removed from the field by uprooting them by hand. Care was taken to avoid breaking the stems. The collected plants were then placed in upright position in a 6-gallon can containing approximately 8 inches of tap water. Twenty-eight days later the plants were inspected for the appearance of fungus growth on the root-and stem-portions at and above the watar line. Microscopic examination of numerous fungus colonies reyealed the presence of Gliocladium roseum and Fusarium species in abundance, Verticillium was not found. Upon splitting ttie stalks, fungi were found growing in the pith cavity slightly above the soil line on the plants and extending 12 to 14 inches upwards. Temporary slide mounts were made of the fungi found in the pith region of all 13 plants. Eleven plants contained Verticillium and 2 bore only Fusarium species. The fungi observed in the pith region appeared to be growing from the xylem into the cavity caused by disintegra­ tion. In areas where the pith had definitely disintegrated, the fungus elements were readily visible when observed through a hand lens, but were not visible where disintegration had not occurred. In order to determine the distance that the fungi had spread longitudinally in the pith cavity, stalks were cut into 1-inch sections that were examined under a binocular microscope after being split. The fungi were not observed in each consecutive stem-piece between the ground line and the stem-tip>. For example; one stalk showed fungus present in a piece taken from the 4-inch level above the soil line and also at the 12-inch level but fungus was not observed in the stem-piece from the 8-inch level. The fungi were consistently present in the more mature parts of the lower stem where disintegration of the pith had occurred. Fungi were not observed in the immature stem-tips where the pith was intact. Isolations of Verticillium and Fusarium were made from the pith region of stalks in the following manner. Stalk sections 4 inches long were surface-sterilized in mer­ curic chloride and cut into 1-inch pieces, which were then placed in a sterile container and removed to a sterile cul­ ture chamber. Stem sections were then sliced longitudi­ nally under aseptic conditions to expose the fungus in the pith cavity. A flamed transfer needle was then stroked over the surface of a potato-dextrose agar slant and touched to the fungus in the pith cavity, A minute portion of fun­ gus adhering to the agar-covered needle tip was transferred to a slant for incubation. Only one transfer was attempted for each 1-inch section. Of 24 attempts, 5 Isolations of Verticillium in pure culture were made from as many plants. Ten attempts gave negative results and 9 produced only Fusarium species. Several branches were examined for the presence of fungus in the pith cavity. Of 16 branches examined by the described method, 3 showed fungus growth in that region. In each case, these were the lowest branches on the stem and therefore the oldest. In no case did the fungus ex­ tend farther than 3 inches from the base of the branch. Generally, the pith in branches was not disintegrated sufficiently to allow space for visible fungus growth. This does not preclude the presence of the fungus in these regions, but large and easily visible colonies were not observed. 155 Examination of pith, taken ftora the vicinity of fungus growth showed that the discoloration was due to disintegra­ tion and to gum-like deposits. In one slide, of a pith portion picked from the stem by needle and mounted in Sar- tory’s solution containing 0.25 percent Orseillin BB, a fungus hypha was observed penetrating cell walls of the pith parenchyma. The hyphae had formed bulb-like protuberance^ on either side of adjacent cell walls* The protuberances were joined by a much reduced hypha1 portion which actually passed through the cell walls. Fifty additional plants, collected from the same area were similarly treated in an effort to determine if the method might have diagnostic value in identifying Verticil­ lium wilt. Ton plants were examined immediately after collection. Verticillium was identified from only one plant. Non-fruiting hyaline hyphae were observed in 4 of the plants. Fusarium was recognized in one pith cavity and was noted to produce hyphae much coarser than those mentioned above. No fungus growth was observed in the remaining four plants. Ten plants were examined at irregular intervals during the 20 days following collection* The first 9 plants ex­ amined produced no recognizable Verticillium from the pith regions. The tenth plant, however, yielded abundant Verti­ cillium. Fifteen plants were examined on the twenty-fourth day with the following results ; 5 positive for Verticillium, 5 producing Fusarium, and 5 "bearing no fungus. The remain­ ing 15 plants were examined on the twenty-ei^th day. Seven produced Verticillium, 4 produced Fusarium. and 4 contained the non-fruiting, very fine, hyaline hyphae believed to belong to Verticillium.

GROWTH COMPARISONS OF ISOLATES ON VARIOUS IVIBDIA The five isolates under study were grown on different media in attempts to further identify them by varialDle growth characteristics. The media upon which the isolates were cultured for comparison included: wort agar, steamed potato plugs, potato-dextrose agar, Czapek’s Solution Agar (as modi­ fied by Raper and Thom, 135), steamed and surface-sterilized cotton stem pieces, and two liquid media, Difco dehydrated wort agar was prepared in the usual manner. Petri plates cbhtaining 20 ml, of the agar were inoculated with approximately equal quantities of each of the fungus isolates by transfer needle and cultured at 23 degrees G, for seven days, Gliocladium grew well on this medium, producing vigorously growing, spreading colonies which were at first pale yellow in color and of granular texture, Conidiophores and conidia were produced in abun­ dance in the dense aerial mycelial growth of older colonies. In contrast, none of the Verticillium strains grew well on wort agar and none produced pseudosclerotia or resting mycelium, Conidiophores and conidia were produced only sparingly by Tipton strain, moderately by McNeal and CMI strains, and not at all by Thomas strain. With the exception of Thomas strain, Verticillium growth was charac­ terized by small, glistening,; circular colonies, somewhat raised in the center, and becoming cerebriform in appearance with age. Thomas strain produced similar colonies that eventually bore a very thin, colorless, aerial mycelium that was distinctly different from the parent culture growing on potato-dextrose agar where dense, fluffy, aerial mycelium was.commonly observed. Figure 27 shows the five isolates on wort agar as contrasted with those growing on potato-dextrose agar. All colonies are of the same age (7 days) and were grown under identical conditions. Cultures grown on potato-dextrose agar have been des­ cribed under "Origin and Description of Cultures” and need not be repeated here, Berkeley et al, (12) suggested the use of potato plugs upon which the fungus was cultured to cause regaining of » resting mycelium* which was lost in repeated transfers on agar media. The fact that the Tipton strain had lost much of its ability to form pseudosclerotia by repeated transfer on artificial media instigated the use of potato plugs. All isolates grew readily on this substrate (Fig. 28), Cultures shown are one week old, Tipton, McNeal, and CMI strains produced appressed gray-white mycelia, the individual hyphae of which were adhering to each other in tïilok strands. Fruiting was profuse in both McNeal arid CMI strains but Tipton strain produced conidia sparingly, and failed to regain its ability to produce pseudosclerotia. Thomas strain formed a dense, fluffy, white aerial mycelium without conidiophores and conidia, as was also typical of this isolate on potato-dextrose agar. None of the Verticil­ lium strains produced pseudosclerotia in great quantity within the 28 days of growth on this medium, McNeal strain appeared pinkish-gray in color at the end of two weeks. The CMI strain also produced pinkish-gray coloration though three weeks were necessary for its appearance. Gliocladium underwent a radical change on potato plugs. Growth of this fungus at one week was coarse and the hyphae were irregular­ ly thickened. Conidia, usually produced in great numbers on other media, were few on this substrate. The conidio­ phores were thickened and stunted and both terminal and intercalary chlamyddspores were formed. At two weeks Gliocladium colonies were generally circular, salmon- colored near the margins, and pale yellow at the center. Growth of the isolates on Gzapek^s Solution Agar was very similar to that occurring on potato-dextrose agar, Tipton strain differed slightly by producing fluffy, white mycelium; all strains grew equally well on this medium.(Pig, 29),. A liquid nutrient normally used in the laboratory of the Department of Plant Pathology for the culture of 159 ' Streptomyces ^rise-ua in tlie production of crude streptomycin was employed as a test medium. The solution consisted of tap water containing 1,0 percent glucose, 0.5 percent peptone and 0,5 percent sodium chloride, adjusted to a pH of 7,0, Ten ml, quantities, sterilized in test tubes, were inoculated with the isolates and incubated at 23 deg, G, for 30 days. All isolates were characterized by the production of flocculent, submerged growth and the early formation of thick, leathery, cone-shaped pellicles over the liquid surface, Thomas and McNeal strains formed numerous pseudosclerotia along the edges of the pellicles in two weeks, though the Tipton strain produced none during the entire period of culture in this medium, CMI strain formed neither resting mycelium nor pseudosclerotia in the' 30-day period but produced numerous conidiophores and conidia. The growth of Tipton strain was similar to that of the CMI strain thou^ it produced fewer conidiophores and conidia. In the case of Gliocladium a thick yellowish-white pellicle bearing granular mycelium was formed, Conidia and conidiophores were produced in abundance, The second nutrient solution had the following formula: sodium nitrate, 3 grams; potassium acid phosphate, 1 gram; potassium chloride, 0,5 grams; magnesium sulfate, 0,5 grams; ferrous iron sulfate, 0,1 grams; and glucose, 20 grams. The constituents were dissolved in 500 ml, of c o m juice and 500 ml. of tap water. This medium was adjusted to 7,0 pH and 160 steam-sterilized. Growth of all isolates in this medium vfas comparable to that occurring in the previously des­ cribed liquid medium. No outstanding variations were noted. Growth was attempted on one-inch portions of steam- and surface-sterilized cotton stems taken from healthy plants grown in the greenhouse. Needle transfers of each isolate were cultured at 25 deg, C, for 30 days. All strains grew well on steam-sterilized stem-pieces; although growth was least abundant in the case of the Tipton strain. At 30 days all mycelial growths were slightly pink-colored when viewed in direct light. Growth in all cases was characteristic of that observed on potato- dextrose agar with the exception of the Thomas strain which, as usual, produced no conidia and on this substrate formed only thickened, torulose, resting mycelium Instead of the numerous pseudosclerotia commonly observed on other media. Growth on surface-sterilized pieces of cotton stem was sparse with the exception of Gliocladium which pro­ duced abundant conidia and also formed thickened, torulose hyphae. The production of pseudosclerotia and resting mycelia by the Verticillium strains was lacking on this substrate. DISCTJSaiON AND CONCLUSIONS

Dissemination of Verticillium wilt of cotton has not yet been explained satisfactorily despite many theories as to the exact means. Rudolph and Harrison (148, p. 858) concluded that seed-transmission of the disease was relative­ ly unimportant; however, in the same paper these workers pointed out the desirability of investigation to further explore the possibilities of seed-dissemination. These workers stated; "The average grower would not recognize or be .concerned with the single, individual plant that might develop the disease among the thousands in his field, assuming the disease to have been introduced on debris, etc. On the contrary, he would ignore it and eventually disk it into the soil along with others at the end of the season, thereby establishing a real focus of infection the following year. The second year there m i ^ t be enough disease pre­ sent to constitute an 'outbreak*." It matters not how wilt becomes introduced into a previously uninfested locality; the statement quoted above will certainly be true, whether introduction is accomplished by debris, soil, water, dust, other host plants, or seed. The important matter is to determine exactly how initial \ introduction takes place so that measures of control may be exercised. There are approximately 65,000 acid-delinted seeds planted in each acre to produce a cotton crop. When 162 one considers tills fact, it ‘becomes readily apparent that the combined efforts of all the investigators, who have sougjit Verticillium in the interior of cotton seeds, has resulted in the testing of only a relatively small number of seeds. It is generally considered that if Verticillium is seed-borne, that only a small number of seeds will carry the fungus. If only 0.1 percent of the seeds planted in a single acre were internally infected there would be 65 chances that infection might occur. The complex of environmental requirements of the parasite and an equally critical set of conditions that must occur simultaneously for the host and the pathogen would in all probability substantially reduce the number of chances for infection. The irregular occurrence of diseased plants tends to con­ firm the reasoning that all of the optimum conditions for the host and the pathogen have been met only in those re­ stricted regions. MeKeen (111) concluded that the epidemio­ logy of "Verticillium wilt is a complex of factors requiring much further analysis. If Verticillium is present in virgin desert soils, as Presley (133) contends, it would seem that Verticillium would be even more widespread than it is now known to be in the state of Arizona, particularly since cotton has been planted continously for a number of years in certain of our agricultural areas. It seems unlikely that a fungus known to prefer moist conditions would exist in the dry. 163 almost barren, soils of the virgin desert. Practically nothing is known about the relationships between Verticillium and possible wild (native) plant hosts commonly found on the desert and Presley admits that why the fungus is present and how it maintains itself is unknown. Wilhelm (183) has pointed out that Verticillium is not essentially a soil in­ habitant which maintains itself saprophytically in competi­ tion with other soil microorganisms but that it should be classed perhaps as a soil invader, introduced with host material that was invaded while the host was alive. The ability of the fungus to exist saprophytically was indi­ cated to a degree by the manner in which the fungi grew on steam-sterilized cotton stem-pieces and non-living seed- coat tissues but generally failed to do so on surface- sterilized pieces which were not completely killed. Presley (133) found no Verticillium in culturing hundreds of roots of a native plant (Larrea tridentata Goville) found grow­ ing in juxtaposition with cotton roots known to be infected with Verticillium. No mention is made of other native plants having been cultured, although it is possible that some do support the fungus though not being susceptible to it. Evidence presented by other workers regarding the seed- borne nature of the disease is admittedly fragmentary. The fact that Verticillium has been isolated from within egg­ plant seed by both Kadow (85) and Richardson (138) and from with.ln tomato seed by Kadow (85) lends support to the theory that possibly cotton seed also may carry the fungus in the seed interior. Taubenhaus (164), who reported find­ ing Verticillium in cotton seeds, thou ^ t that "one year ^ the infection in the interior of delinted seed is present and another year it may not be." The variability of the causal organism in its reactions under relatively constant conditions lends strength to this theory. It seems entirely possible that the optimum set of conditions for the growth of the fungus through the vascular system of the plant into the seeds may occur in only very few instances. Llosa (105) reported having recovered -Verticillium from the interior of seeds and specified that these seeds from which the fungus was isolated were taken from late, immature bolls from severely infected plants. This, in itself, is rather a strict set of requirements and would limit the number of possible infections. Llosa specified that the infected seeds were borne in the discolored portions of the locks nearest the receptacle. Discoloration of locks as mentioned by Llosa has not been observed occurring in naturally in­ fected plants according to Rudolph and Harrison (148). Also, the writer has found none. The inoculation of pedicels and bolls in the field by the writer produced a condition comparable to that described by Llosa in that the lower parts of locks were discolored at first yellow-brown but later appeared gray-black. It was also noticeable that 16 5 seeds in locks that were visibly discolored failed to develop completely. It was evident from the inoculation of bolls through the locules that the fungus required moist conditions for continued growth. As soon as the boll opened, either because of maturing or because of the injury to the boll, fungus growth stopped abruptly as evidenced by the sharply delimited discoloration of the lock, Rudolph and Harrison (148) found that sterile, water-soaked lint would support growth of Verticillium but that dry fibers did not. Growth on and in lint has been demonstrated in inoculated bolls in the field where the natural plant juices, with which the lint is saturated, probably constitutes a much more favorable medium than lint saturated with distilled water. Boll-and pedicel- inoculations both showed that all tissues of the boll are capable of supporting growth of the fungus while moist but when dried, fungus growth ceases. The fact that the fungus grew from only 25 of 4,805 seeds cultured (0.47 per­ cent) six-months after collection indicates that a small number of seeds were invaded although many of the seeds were taken from locks that showed the previously described discoloration and invasion by the fungus. It is also notable that those seeds from which the fungus was recovered, with only one exception, were those taken from locks definitely bearing Verticillium growth, A possible explana­ tion for the lack of discolored locks occurring in naturally 166 infected bolls might be that Verticilli-um is known to be confined to the vascular system and has shown little tendency for lateral growth from these regions except when the plant is dead. However, in a very severe in­ fection and with optimum environmental conditions, in conjunction with the required host-pathogen relationships, penetration of the inner lining of the locule from the sub-carpellary complex or the boll-core may be possible. Culture of 410 young bolls failed to show penetration of these organs by the parasite. Inoculation of bolls both throu^ the pedicel and carpel has shown that penetration of septa by Verticillium had occurred. It seems reasonable therefore, that the fungus may well penetrate the similar tissue in the pits occurring in the locule base. Injection of dye into boll pedicels indicated that inoculum was not being forced through the pits into the locule, but that penetration of that part was accomplished by the fungus growing through it after having been placed in quantity into the receptacle. Rudolph and Harrison (148) were able to recover Verticillium from 150 boll receptacles although 3,371 were cultured, and of the 150 only 2 showed infection of the core at the base of the placental column. It should be noted that the bolls were described as mature and unopened. This circumstance prompts the thou^t that had the bolls been left on the plant, where they would have remained moist 167 inside longer than when stored in a dry situation, further penetration of the boll (at least in the 2 cases) might have occurred. In order to determine whether the fungus, after reach­ ing seeds, would remain alive there and attack the seedling, it was necessary to inoculate seeds. It is evident that the fungus can survive in seeds after having been placed there artificially for periods in excess of 17 months and can be recovered from those seeds when cultured. Furthermore, inoculated seeds can and do germinate, A point that may be regarded as questionable is that the inoculum may not have been placed by artificial means into the same tissues as might be invaded in naturally infected seeds as found by Taubenhaus (164) and LIosa (105), One outstanding feature observed is the apparent in­ ability of the fungus to invade the uninjured embryo. In repeated cases Verticlllium was observed to grow in wounds in the cotyledons without causing disease of the developing plant. In other instances mycelium was found growing on the cotyledonary tissues without giving indication of active penetration. Histological sections of inoculated seeds showed the fungus had grown in the spaces in the folds of the cotyledons but evidently lacked the ability to penetrate the epidermis, except in a very few instances. Invasion of the seed coat and the fringe tissue was markedly consistent following inoculation by all methods. 168 Tills fact was considered significant in that the vascular system in the seed may be traced through the funiculus and raphe to the chalaza from which the system branches into the outer integument. In the chalazal region, where the vascular system is concentrated, fungus growth from cultured inocula­

ted seeds was common. If natural infection of seed does take place it seems likely that these are the tissues in which the fungus would be found. This suggests that since r. the vascular system of the host plant does not enter the embryo, the developing seedling could throw off the infested seed coat an^ grow to be a healthy plant with the cast-off seed coat acting only as soil inoculum for an adjacent plant or one planted later. With acid-delinted seed the deposition of the cast-off seed coat on the soil surface is of frequent occurrence, and thus the source of infection is carried away from the most likely point of infection of the seedling, the root area. With normal cultural practices the cast-off seed coat could be carried down into the soil only to contact a future actively growing root; and with all of the required conditions of infection at an optimum, infection may take place. Rudolph and Harrison (148) also questioned the possi­ bility that infected seeds would produce diseased plants. The research just completed failed to show that such in­ fection would occur, either in normal field soil or in sterilized soil in the greenhouse. Failure to obtain 169 infected, seedlings and mature plants may possibly be due to the fact that all of the environmental requirements were not met and with repetition of the experiments using larger numbers of seeds under differing environmental conditions infection of a few seedlings might be obtained. Further work is necessary and will be continued along these lines to determine whether the inoculum introduced into the soil by infected seeds would be sufficient to result in attack of a second crop of cotton planted in the same soil. It must be pointed out that new plots were used for each of the field plantings during the investigation. Investigations with Gliocladium roseum were continued after its true identity was known because of its similarity to Verticillium. This resemblance may have contributed to the number of conflicting reports found in the literature regarding Verticillium, Except to one engaged primarily in taxbnomic study of fungi, G, roseum mi^t easily be mistaken for Verticillium. To illustrate this point: mass transfers of G, roseum from a single parent culture were sent to five different agencies, three of which maintained mycologists for the purpose of identifying fungi. Two of the reports received from these three agencies agreed in the identification of the isolate as Gliocladium roseum Bainier. The third report,however, stated that the fungus was a Verticillium, though apparently not V. albo-atrum. One of the two remaining agencies regarded the isolate as a form of V. albo-atrum Relnke and Berthold "since it appeared to be identical in morphological characters". The fifth report also recognized the similarity by stating that the culture was believed to be a species of Verticillium thou^ it was not thought to be Verticillium albo-atrum. Tehon and Jacobs (166) described Verticillium rhizo- phagum n. sp., a fungus that has a striking resemblance to Gliocladium roseum. Dodge (57) described a similar organ­ ism, Verticillium Buxi, occurring on Buxus sp., but stated that V. Buxi was not like G, roseum. Raper and Thom (135) cite workers of the Department of Agriculture in Washington, D, G ., who have reported extensive losses in Buxus due to G. roseum althou^ this fungus is generally regarded as a saprophyte occurring on decaying plant parts. - The resemblances between Verticillium and Gliocladium isolates were considered sufficient to include the latter in the various experiments covered by this work. Gliocladium was not found to be pathogenic to cotton when incorporated into the soil. Slight discolorations were noted in inocula­ ted stems but disease was not caused. When introduced into bolls and pedicels by hypodermic injection, Gliocladium proliferated and caused damage to the carpels, lint, and seeds, and therefore might possibly be regarded as a "wound" parasite. Wounding of seeds in seed-inoculation by Methods A and B reduced the rate of germination of those planted in the field, particularly those seeds of the long-staple varieties. It,is of interest that the seeds inocnlated ■with Gliocladi-um in the 1951 field planting produced fewer emerging seedlings than did the seeds inoculated with the two Verticillium strains. This is further indication of the ability of Gliocladium to behave as a wound parasite. The writer observed that the CHI strain of Verticillium, an isolate forming only bro’vm, torulose resting mycelium and never producing pseudosolerotia, remained true to type throughout the experiments and after numerous transfers upon artificial media. Even thou^ this strain was inocu­ lated into living plants, where definite pathogenesis was observed, no deviations from parent type were noted when reisolations were made. This fact lends some strength to the contention that V, albo-atrum and V, dahliae are differ­ ent species; however, Thomas strain, which typically formed numerous pseudosclerotia, produced only torulose resting mycelium in 50 days on steam-sterilized cotton stem-pieces, Presley (133) has pointed out that monoconidial isolates of Verticillium showed a wide range of variability, includ­ ing the production of only dark, septate hyphae by certain variants. Other strains or variants demonstrated relative constancy, such as the appressed mycelial type which seldom produced variants. The Tipton strain was such an isolate and retained its appressed mycelial form with consistency. This investigation has produced no results that would warrant findings other than agreement with Presley (133) 1V2 and otliera, that all of the herein designated strains of Verticillium should be regarded as variants of the species albo-atrum. Gliocladium was easily separated from the Verticillium strains by culturing the isolates on wort agar. Since Gliocladium grew well while Verticillium did not, this procedure may be used diagnostically. Pathogenicity tests have shown that the influence of the host and environment must be given much consideration. All of the Verticillium isolates were pathogenic to eggplant, all except Thomas strain were pathogenic to okra, but none of the tomato inoculations were considered successful under the conditions of the experiment. All strains of Verticillium were pathogenic to cotton under the same conditions. The inoculation of succulent plant stems with aqueous suspensions of the several strains failed to show greater pathogenicity generally attributed to the pseudosclerotial types. The results were inconclusive in cotton plants grown in soil to which infested oats had been added. Considering only seedling wilt at two weeks, McNeal and Thomas strains (both pseudosclerotial types) appeared most virulent. If seedling deaths are the main criterion, little difference occurred. By accepting the more positive indications, foliar symptoms and vascular discoloration, GMI strain seemed most capable of causing disease. The small number of infections occur­ ring at the end of the ten-week period probably is the 173. result of having added insufficient inoculum to the soil, together with inadequate control of environmental factors, Williams et al. (188) concluded that definite control of factors such as temperature and humidity were necessary for reliable results. Cotton grew poorly under greenhouse conditions and was generally etiolated and chlorotic. This last factor may well have influenced the susceptibility of the plants by increasing resistance to the disease. Observations of the influence of nitrogen upon the expression of symptoms suggested a positive relationship. In pot tests where the nitrogen supply was depleted as the plants grew, foliar symptom-expression disappeared com­ pletely, When nitrogen in the form of commercial ferti­ lizer was added foliar symptoms reappeared. Loss of foliar symptoms was repeated with depletion of the added nitrogen. The isolation of Verticillium from the pith region of dead, mature, moist cotton stalks may be of use, particular­ ly in obtaining pure cultures of the organism. The procedure requires approximately 28 days’ preparatory time but is not particularly tedious. The method does afford definite iden­ tification of the disease without usual cultural techniques unless isolation of the fungus is desired. Occurrence of the organism in the pith region of dead stalks standing in the field should be further investigated inasmuch as Blank and Leyendecker (16) have shown that debris from infected cotton plants is capable of spreading the disease. SUMMARY

In view of the fact that dissemination of Verticillium wilt of cotton has not yet been satisfactorily explained it was deemed desirable to further explore the possibilities of seed-transmission- Over a 3-year period approximately 11,500 seeds of several cotton varieties were inoculated by three methods with 4 isolates of Verticillium and 1 of G-liocladium. Verticillium has been shown capable of living in the interior of viable cotton seeds for more than 17 months, Verticillium-infested seeds can and do germinate. The por­ tions of the inoculated seeds that appear to be Invaded by the fungus are the seed coat and the fringe tissue. In the chalazal region, where the vascular system of a seed is concentrated, fungus growth from cultured inoculated seeds was common. Cultural and histological studies have shown that the embryo is not attacked unless wounded. Three described methods of inoculation of seeds were successful in laboratory testa, but 8,330 inoculated seeds planted in either sterile or field soil did not produce plants which were diseased at maturity. It is suggested that the infested seed coat may constitute a source of inoculum for the infection of an adjacent plant or one planted later. Further investigation is to be conducted 175 to determine this point & Over 700 injections of inoculum into various parts of field- and greenhouse-gro^ plants were made. All parts of the hell appeared to act as a favorable medium for the growth of Verticillium. The only barrier to seed infection appears to be a lack of moisture in the maturing.boll. Moisture has been shown to be a prerequisite for growth of the fungus. Althou^ 410 young bolls from infected plants were cultured with negative results, the fact-that all parts of a boll will support growth of the fungus in­ creases the likelihood that seeds may act as a dissemina­ ting agent of the disease. Verticillium was obtained from 25 (0.62 percent) of 3,688 acid-delinted and surface-sterilized seeds which were cultured approximately 6 months after harvest. ' "À1- thou^ the percentage of infected seeds was found to be low, possible centers of field infections from'which fur­ ther spread may take placefare indicated by this study. The capability of Verticillium to penetrate septa in­ dicates a possibility that the fungus can enter the boll from the sub-carpellary complex by way of the pit at the base of each locule. Growth of Verticillium variants on several artificial media revealed no outstanding differences between the isolates. The capacity of Verticillium to exist sapro­ phytic ally was indicated by successful culture of the 176 fungus upon non-living plant materials. ■ Pathogenicity tests have shown that relative patho­ genicity is difficult to determine without adequate con­ trol of environmental factors and that expression of symptoms is variable with the mode of introduction of the pathogen to the host. Both species, V. albo-atrum and V. dahliae, if such specific differentiation is warranted, are pathogenic to cotton. No correlations between morpho­ logical type and pathogenicity could be found. Observa­ tions of the influence of nitrogen upon the expression of foliar symptoms suggested a positive relationship. G-liocladium roseum Bainier. a fungus that may be mistaken for Verticillium species, has been found to be non-pathogenic to cotton except perhaps in the relation of a "wound" parasite. Gliocladium roseum grew well on wort agar while Verticillium Isolates did not; this may constitute a cultural means of distinguishing isolates of the two genera, Verticillium has been found growing in the pith region of dead, mature stalks, A method of isolating the fungus from pith into pure culture has been suggested. Growth of Verticillium in stem-pith may have diagnostic value in the identification of Verticillium wilt of cotton. LITERATURE CITED

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105, LI08a, T, InvëstigaclGhes^reférèritës al ’’wilt” del algodonero y nuevo metodo para aislar bongos del tipb Verticillium o Püsarium d e .. plantas atâcadàs por èl "wilt" del algodbriero. Bol, Estac, exp, agric. La Molina, No, 13, 1938. 106, Ludbrbok, W. V, Pâthogéhicitÿ'and. ényironal ' studies on Verticilliüm hadromycosis. Phyto- path. 23; 117-154. 1933, 107, Mackié, W, W. Blàckeyè bèans in Galiforhia, ; Calif, Agr, Exp, Sta. Bull, 696, 1946, 108, Marchai, E, .Observations et recherchés gffëctuees a la Station dë Phytopâthôlogië de 1^ Etat ' pendant 1»année 1939-1940. Bull. Ihst,‘agron. Gemblbüx, 9; 1-15, 1940; 10; 5-10, 1941. (Abstract Tn R. A, M. 24:~¥91. 1945.). 109, Martin, W.' H. New Jersey Agricultural Experî- ' mëht Statiôh. 52nd, Ann, Rept. for yèar end­ ing June 30, 1931, pp. 47-52, 307-316. 110, McKay/ M. B . Purthér stüdiës 'of potato wilt ' ' caused by Verticillium albo-atrum. Jour, Agr, Res. (TJ. SVl 32; 437-470. ''l'9'2g." " 111, McKëén, 0, D. A study of some factors affect­ ing the' pathogenicity of Verticillium albo- atrum R, & B, Cahad. Jour, Res., Sect, "C', S X i 95-117. 1943, 112. Meër, J. H, H, van der. Vërticilliüm-wilt of ... herbaceous and woody plants,‘ Med. Landouwhoogesch, (Wageningen) 28; 1-82. 1925, 113, Miller, P, A. Diseases of brhamehtai plants in Southern California, Phytopath, 28: 672, 1938. 187 114. Miller, F, A. Hôtes on diseases of ornamental plants in Southern California, TJ, S, Dept, Agr., Pl. Dis. Reptr. 24; 219-222. 1940. 115. Miller, P. R., and Nellie Nance. Preliminary estimates of acreages of crop lands in the United States in­ fested with some organisms causing plant diseases. U, S. Dept. Agr., PI. Dis, Reptr, Suppl. 185; 207- 252, 1949. 116. Miles, D, E, Verticillium wilt of cotton in Greece, Phytopath. 558-559, 1934. 117. . The Verticillium wilt disease of cotton. Phytopath. 2^i 972-973, 1935,

118. 9 and T, D, Persons. Verticillium wilt of cotton in Mississippi, Phytopath, 22; 767-773, 1932. 119. Moore, W, C, Seed-home diseases, Ann, Appl, Biol, 55; 228-231. 1946. 120. Mujica, P. Patogenicidad de algunas cepas del Verticillium albo-atrum Rei. y Berth, Bol. Sanid. veg,, Santiago, 1; ^-^0, 1941. 121. Nelson, R. The specific pathogenesis of the Verticillium that causes wilt of peppermint. Phytopath.57; 17, 1947. 122. ______, Development of varieties of spearmint resistant to Verticillium wilt and to rust. Phytopath, 20, 1950, 123. ______. Verticillium wilt of peppermint. Mich, Agr. Exp, Sta. Tech, Bull, 221. 1950, 124. Nielsen, L, W. Verticillium wilt of potatoes in Idaho. Idaho Agr. Exp, Sta. Res. Bull, 15, 1948, 125. Oknina, Mme. E . Z . The Verticillium disease of cotton. Trans. Timiryazeff, Inst, PI. Physiol, 2; 83-115, 1937. (Abstract in R. A. M. 3^; 814. 1938.). 126. Osmun, A. V. Mass, Agr. Exp. Sta, Bull. 293. pp. 15-21, 1933. 127. . Rept. Mass. Agr. Exp. Sta., 1934, pp. 23-27. Ï935. 188 128. OvGharov, K, On th.e ferment of pathogenic fungi causing the splitting off of urea from protein, C. R. Acad. Sci. U. R. S. S., (N.. S.), 20; 577- 380. 1938. (Abstract in R. A. M. 18; 198. 1939.). 129. Pan Union Academy of Agricultural Sciences, Moscow, 24 pp., 1940. (Pests and diseases of cotton and lucerne and their control.) (Abstract in R. A. M. £7; 475. 1948.). 130. Patel, M. K . , I. M. Qureshi, and V. P. Bhide. First record of Verticillium wilt in India. Indian Phyto- path. 2; 245-246. 1949. 131. Presley, J. T. Saltants from a monosporic culture of Verticillium albo-atrum. Phytopath. £1; 1135-1139. 1941. 132. ______. Report of sub-committee chairman on Verticillium wilt for 1948. Phytopath. £9; 499. 1949. 133. , ______. Verticillium wilt of cotton with par- ticular emphasis on variation of the causal organism. Phytopath. 40; 497-511. 1950. 134. Rada, G. G. Departamento de Fitopatologia. Memoria del Jefe de la Seccion Fitopatologia. Mem Est. exp, agric. La.Molina. 11a, pp. 233-284. 1939. 135. Raper, K. B., and C. Thom. A Manual of the Penicillia, 875 pp. The Williams & Wilkins Co., Baltimore. 1949. 136. Reinke, J., and G. Berthold. Die Zersetzung der Kartoffel durch Pilze. Untersuch. Bot. Lab. Univ. Gbttingen. 1; 1-100. Vfiegandt, Hempel, und Parey. Berlin. 1879. 137. Report of the Chief of the Bureau of Plant Industry, Soils, and Agricultural Engineering, Agricultural Research Administration, 38 pp., 1945. 138. Richardson, J. K. Eggplant wilt. Sci. Agric. 14; 120-130. 1933. 139. Richter, E., and M. Klinkowski, Wirtelpllze-Welkekrank- heit an Luzerne und Esparsette (Erreger; Verticillium albo-atrum Rke et Berth.). NachrBl. dtsch. PTÏScHÜTënst. 18; 57-58. 1938. (Abstract in R. A. M. 17: 754-755. 1^38.). 189 140, Ridgway, R, Color Standards and Color Nomenclature, 53 colored plates, Washington, D. C, 1912. 141, Roberts, F, M. Factors influencing infection of "the tomato by Verticillium albo-atrum, Ann, Appl, Biol, 30: 327-31, 1943. 142, ______, Factors influencing infection of the tomato by Verticillium albo-atrum. II, Ann, Appl, Biol, 191-193, 1944. 143, Rudolph, B, A, A preliminary report of a digest of the -world’s literature on hadromycosis due to Verticillium spp. Phytopath, 1^; 1138, 1929. 144, ______, Verticillium Hadromycosis. Hilgardia, 5s 197-361. 1931, . “ 145, . An experiment to determine the sus- ceptibility of flax to Verticilliosis, Phytopath. 25; 892. 1934- 146, , The unimportance of tomato seed in the dissemination of Verticillium wilt in California. Phytopath. 622-630, 1944, 147, , , and G, J, Harrison, Attempts to con­ trol Verticillium wilt of cotton and breeding for resistance. Phytopath, 753. 1939, 148, ______and . The unimportance of cotton seed In the dissemina-bion of Verticillium wilt in California. Phytopath. 849-860. 1944, 149, , a n d ______The invasion of the Internal structure of cotton seed by certain Fusaria, Phytopath, 542-548, 1945, 150, Ruggieri, G, Possibili casi di tracheoverticillosi fra gli Agrumi, Ital, agric,, 1946, 8, 4pp,, 1946, (Abstract in R, A, M, 317, 1948,), 151, Sarejanni, J. A, La verticilliose du Coton en Grèce, Ann. Inst, phytopath, Benaki, 2; 79-85, 1936, 152, Schaible, L,, 0, S, Cannon, and V, Waddoups. In­ heritance of resistance to Verticillium wilt in a tomato cross, Phytopath, 986-990, 1951, 153, Schneider, H, Verticillium wilt of Guayule, Phytopath, ^ s 936, 1944. 154, Shapovalov, M., and J, M, Lesley. A tomato resistant to two wilts, Phytopath. 27; 955, 1937, 1 9 0

155. Sherbakoff, C. D, Verticilli-um wilt of cotton, Pliyto- path. 19: 94, 1929. 156. Simpson, D. M., C. L, Adams, and G. M. Stone. Anatomi­ cal structure of the cottonseed coat as related to problems of germination. TJ. S. Dept. Agr., Tech, Bull. 734. Nov., 1940. 157. Smith, L. and C. Wilson, Verticillium wilt of cotton in Alabama. IT. S. Dept. Agr., PI. Dis. Reptr. 35; 165. 1951. J 158. Snyder, W. C. Seed dissemination in Fusarium wilt of , Phytopath. 253-257, 1932. 159. ______, H. N, Hansen, and S. Wilhelm. New hosts of Verticillium albo-atrum. TJ. S. Dept. Agr.. PI. Dis. heptr. 26-27. 1^50. 160. Suchorukoff, K., and B. Strogonoff. The activators of peroxidase in sick plants. C. R. Acad. Sci. TJ. R. S. S., (N. S.), 15: 563-565. 1937. (Abstract in R. A. M. 15: 808-80WT 1937.). 161. Sukhorukoff, K. T. A study of the characters indi­ cating resistance of cotton varieties to wilt and gummosis. Trans. Timiryazeff Inst, PI. Physiol. 2; 117-137. 1937. (Abstract in H. A. 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Effect of various soil amendments on the inoculum potential of the Verticillium wilt fungus Phytopath. 684-690. 1951. 183.______. Is Verticillium albo-atrum a soil invader or a soil inhabitant? Phytopath, 944-945. 1951. 184.______. Verticillium wilt and black root of straw­ . Calif. Agric. 6z 9, 14. 1952. 185.______, and H. E. Thomas. Verticillium wilt of bramble fruits with special reference to ursinus derivatives. Phytopath. 40; 1103-lliÛ. TW5Ô:— — 186. Williams, P. H., Enid Oyler, H. L. White, 0. C, Ains­ worth, and I, W. Selman. Plant Diseases. Rep. exp. Res. Sta, Cheshunt, 1939, pp. 28-38, 1940. 187.______, Enid Sheard, I. W. Belman, and 0. Owen. Plant Diseases. Rep. exp. Res. Sta, Ches- hunt, 1944, pp. 21-31, 1945. (Abstract in R. A. M. 25; 29U-291. 1946.). 188.______, "W. H. Read, and I. W. Selman. Plant Diseases. Rep. exp. Sta. Cheshunt, 1943, pp. 28-52, 1944. 189. , I. W. Sel­ man, and E. Grossbard. Plant Diseases. Rep. exp. Res. Sta. Che shunt, 1945, pp. 25-65, 1946. (Abstract in R. A. M. 26; 83-184. 1947.). 193 190* Wollenweber, H, W* Die Wirtelpllz-»WeIkekrankbe 11 (Verticllllose) von Ulme. Aliorn "und Dinde nsw, Arb. Biol* Reicbsanst, fnr Land- und Porstwirtsch, 1 7 ; 273-299. 1929. (Abstract in R. A. M. 9; 6. 1930.), 191, Zeller, S. M, Verticillium wilt of cane fruits. Oregon Agr. Exp. Sta. Bull. 344. 1936. 192. Zentraeyer, G. A. Verticillium wilt of . Phytopath. 59: 26, 1949, APPENDIX r . ? '^* h

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Pig, 1. Long, slender conldioplaores of Gllocladlum roseutn with apiculate conidia, (Approx. X20by. Fig. 2. Typical colonies of the fungi under 3tudy°grown on potato-dextrose agar for 7 days at 25 deg. C. From left to right: top, Tipton, Thomas; center, Mcîleal, CMI (all Verticillium strains); and bottom, Gliocladium. M l

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Fig. 3. Equipment used for inocu­ lation of seeds by Method A; holding board with receptacles for seeds, hypo­ dermic syringe, and shortened, re­ sharpened syringe needle. Pig. 4. Colonies of Tipton strain of Verticillinm growing from the wound-site and raicropyle of SxP variety seeds inoculated by Method A. (Approx. 8 months after inoculation).

Fig. 5. Tipton strain of Verticillium growing from germinated seeds inoculated 8 months before culture on agar. Pig. 6. Photomicrograph of Tipton strain of Verticillium recovered from hypodermic ally inoculated seed. Arrow points to conidiophore. (Approx. X830). ^ 2 ' «

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Fig. 7, Portion of a longitudinal section of a Verticillium-infected seed inoculated by Method A, Fungus may be seen in the cotyledon-tissue as well as in the spaces of the cotyledonary folds. Section stained with Safranin 0-Heidenhain*s iron-hematoxylin. (Approx. X200) Pig* 8* Camera lueIda drawing of Verticillium hypha penetrating parenohyma cell walls. Partially disintegrated aleurone grains and asteroid-sliaped nuclei are shown. (Approx. X2550). Fig. 9. A ten-week-old cotton plant of the 1517 RB variety infected with the CMI strain of Verticillium. Lower leaves are wilted, curled, and bear large, irregular, necrotic patches. Pig. 10. Tipton strain ______Verticillium_ growing from (w) the wound-site in the cotyledon and from (s) the fringe tissue inside the cast-off seed coat. Pig. 11. Sharply delimited lesion bearing Verticillium (Tipton strain) on the cotyledon of the seedling at the right, On the left, Verticillium is seen growing from the edge of a wounded cotyledon and from the cast-off seed coat. /

Fig. 12, Drilling apparatus used for prepara­ tion or seeds for inoculation by Method B, Hand drill (a) is attached to tripod (b) leg by means of clamps. Drill-point extends through hole in drill board (c). Seed is pressed against spring on board to make contact with drill point in seed­ shaped cavity hollowed from the upper surface of the board. Pig. 13. Verticillium growing from seeds inoculated by Method C. Mycelium most evident on (a) and in (b) seed coats and from portions of cotyledons (c) injured in attempt to shed the clinging seed coat. Pig. 14. Verticilli-um colonies on potato-dextrose agar to which the fungus was carried from the interior of seeds hy the seedlings. Two seedlings (indicated hy arrows) in the lower portion of the photograph are being invaded and killed by growth of the fungus from the clinging seed coats. Pig, 15, Growth of CMI strain of Verticillium from the inoculation wound-site and from the chalazal end of the germinated seed.

Fig, 16, Cotyledons wounded in inoculation by Method B, Arrows point to Verticillium colonies (Thomas strain) at wound-sites, %

Pig, 17, Hypodermically inoculated cotton stems showing vascular discolora­ tion. From left to right; inoculated with Gliocladium, CMI strain, and McNeal strain! Arrows point to inoculation site. Little discoloration occurred in the stem receiving Gliocladium. Photographed two months after inoculai:ion. Fig. 18. Modified kitchen strainer used for surface sterilizing seeds. Pig, 19, Growth of Gliocladium from pedicel and receptacle of boll 14 days after being inoculated through the pedicel. Pig, 20. Locks of lint from bolls inoculated through, the pedicel. From left to right; Gliocladium; Thomas, Mc­ Neal, Tipton, and CMI strains of Verticillium. Pig, 21. a) Pseudosclerotium of McNeal strain of Verticillium in cotton fiber-lumen. (Approx. X865). b) Lower 'magnification of liyphae and pseudosclerotia of McNeal strain in and on lint fibers. (Approx. X200) a

_ DO a) Vertic ill i-uni of the CMI strain invading n^r.1- ffber * (Approx. Xy8H)T" b) Resting mycelium of CMI strain l^lnd on lint fibers. (Approx. X200). #

Pis. 23. Gliocladium liypîiae have penetrated a cotton fiber and may be observed at (1) in the lumen, and at (w) in the fiber wall. (Approx. X190). r

Pig. 2 4 . ’JVhite mycelium of Gliocladium on se p ta and placental column of cotton boll. Inoculum was injected into discolored lock. (Approximately natural siz e ). Pig. 25. Verticillium-wllted cotton plants 11 days after hypodermic inoculation. Plant in pot 3, McUeal strain; pot 4, CMI strain; and pot 5, Tipton strain. Fig. 26. Cotton plants 13 days after hypodermic inoculation. Plant in pot 2, beginning to wilt, was inoculated with Thomas strain; plants in pots 1 and 6 were inoculated with sterile water and Gliocladium, respectively, and show no wilting. 9-VlorT

Pip;. 27. Growth, of Vertlcillium and Gliocladium isolates on wort agar after o days ; 1, T ip ton ‘strain; 2, McNealj 3, Gliocladium; 4, Thomas; and 5, Cm I. Fig. 28. One-’week-old cultures of Vertlcillium. and Gliocladium isolates on potato plugs. Prom left to right : Tipton, Thomas, McNeal, CMI, and Gliocladium. Fig. 29. Eight-day-old cult-ures on Gzapek’a Solution Agar. From left to right: top, McNeal, CMI; center, Tipton, Thomas; and at bottom, Gliocladium.