by
A
submitted to
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
June 1960

TPPEffi.EDT
ol
Lr
SaooL

ACKNOWLEDGEMENTS
I am grateful to Dr. Chester E. Horner, for suggest­ ing this problem and for his invaluable advice and coun­ sel throughout the course of the investigation and in the preparation of the thesis. The valuable criticism of the manuscript made by Dr. Lewis F. Roth is also appreciated.
I would like to thank in this thesis.
Mr. H. H. Millsap for his as­ sistance in making the photographic duplications included
The financial assistance provided by major users, buyers and growers of peppermint oil through the Oregon
Essential Oil Growers League is also gratefully appreciated.

TABLB OF CONTENTS
INTRODUCTION • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •
Page
1
REV lEW OF LITERA TUBE • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 3
II.ETHODS AND IIATEllIALS . • • • . . • • • • • • • • • • • • • • . • • . . • • • • • . 1 4
Culture. . • • • . • • • • • • • • • • . • • • • • • • • • • • • • • . • • • • • • . • • • 1 4 lledia............................................ 1 4
Preparation of inoculum.......................... 1 4
Soil... . • • • . • . . . . . • . • • • . • • . . . • • • . • . . . • . • • . . . . • . . . 16
Soil moisture.... . • • . . . . • . . . . • • • • . . . • . . . . . . . • • . • . 16
Soil temperature control......................... 18
Saapling. • • • . • • • • • • • • • . . • . • • • • • • • • • • • • • • • • • • • • • • • 18
Population assay by soil dilution................ 19
Assay by indicator plants........................ 22
THE EFFECT OF SOIL MOISTURE AND TEMPERATURE
ON SURVIVAL OF VERTICILLIUM KICROSCLEROTIA.......... 2 4
Soil temperature tank experiaent................. 2 4
Teaperature cabinet experiments.................. 32
1) Preliminary experiment...................... 32
2) Final teaperature cabinet experiment........ 37
Effect of high teaperature on survival........... 4 0
DISCUSSION.......................................... 52
B IBLIOORAPBY. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 60

S
Pa g e
1. Survival of an initial 2.3 million Verticillium microsclerotia per cc over a six month period in soil held at 5 levels of moisture and 4 levels of temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2. Percentage reduction of Verticillium microscle­ rotia in soil after a six month period at 5 levels of moisture and 4 levels of temperature ••••••.•••• 26
3. Isolation results from the temperature tank ex­ periment showin g effect of moisture and tempera­ ture on the population of Verticillium mieroscle­ rotia in soil as assayed by indicator plants.
Soil samples were held for si x months at indicated moisture and temperature before assayed ••••••••••• 27
4 . Effect of different levels of soil moisture at s•
Con survival of Verticillium micros c lerotia •••••• 33
5. Effect of different levels of soil moisture at 10•
Con survival of Verticillium microsclerotia •••••• 33
6. Effect of different levels of s o il moisture at 15°
Con survival of Verticillium microsclerotia •••••• 33
1. Effect of different levels of soil moisture at 20•
Con survival of Verticillium microsclerotia •••••• 34
8 . Effect of different levels of soil moisture at 25°
Con survival of Verticillium microsclerotia •••••• 34
9 . Effect of different levels of soil moisture at 30 °
Con survival of Verticillium microsclerotia •••••• 3 4
1 0 . Percentage reduction of Verticillium microscle­ rotia after a six month period in soil held at various levels of moisture and temperature •••••••• 35
11. Isolation results from the temperature cabinet e x ­ periment showing effect of mois t ure and tempera­ ture on the population of Verticillium microscle­ rotia in soil as assa y ed by indicator plants. Soil samples were held for six months at indicated moisture and temperature before assayed ••••.•••••• 3 8

Page
12. Percentage reduction of Verticillium microscle­ rotia after a six month period in soil held at various levels of moisture and temperature •••••••• 4 7
13. Effect of different levels of moisture at 30 ° and
4 0• Con survival of Verticillium microsclerotia •• 49

LIST OF FIGURES
Page
1. Comparison of growth of peppermint plants after two aonths in Verticilliua-infested soils at three levels of moisture. The soils had been incubated for six months at 10• C prior to plant iDg. . • . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . • . 28
2. Comparison of growth of pepperaint plants after two aonths in Verticilliua-infested soils at three levels of aoisture. The soils bad been incubated for six aontbs at 20° C prior to planting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3. Coaparison of growth of peppermint plants after two months in Verticilliua-infested soils at three levels of aoisture. The soils bad been incubated for six aontbs at 30• C prior to planting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4 . Comparison of growth of pepperaint plants after two aonths in Verticillium-infested soils at three levels of moisture. The soils bad been incubated for six aontbs at 4 0° C prior to planting........... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
5. Effect of five levels of aoisture on survival of
Verticilliua aicrosclerotia in soil held at 5• C for six aooibs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
6. Effect of five levels of aoisture on survival of
Verticilliua aicrosclerotia in soil held at 10•
C for six aontbs................................ 4 2
7. Effect of five levels of aoisture on survival of
Verticilliua aicrosclerotia in soil held at 15•
C for six aontbs..... • • • • • • • • • . • • • • • . • • • • • • • • • . • 43
8 . Effect of five levels of aoisture on survival of
Verticilliua aicrosclerotia in soil held at 20°
C for sixd aontbs............... . . . . . . . . . . . . . . . . . 4
9 . Effect of five levels of moisture on survival of
Verticilliua aicrosclerotia in soil b~ld at 25•
C for alx •ouths......... . . . . . . . . . . . . . . . . . . . . . . . 4 5
10. Effect of five levels of aoisture on survival of
Verticilliua aicrosclerotia in soil held at 30•
C for six aoatbs..... . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Page
11. Effect of three levels of moisture oo survival of Verticillium microsclerotia in soil held at
30• C for 35 days... . . . . . . . . . . . . . . . . . . . . . . . . . .
12. Effect of three levels of aoisture oo survival of Verticillium microsclerotia in soil held at
40 ° C lor 35 d ays... . . . . . . . . . . . . . . . . . . . . . . . . . .
50
51

THE EFFECT OF SOIL MOISTURE AND TEMPERATURE ON
SURVIVAL OF VERTICILLIUM ICROSCLEROTIA
INTRODUCTION
The fungus Verticillium albo-atrum Reinke and Berthold causes a wilt disease of more than 20 0 species of plants, includin g many of g reat a g ricultural importance. Investi­ gations by Nelson (25, p . 65 69) describe the g enerally ac­ cepted life cycle of V. albo-atrum. Accordin g to his find­ in g s infection occurs throu g h the roots and then the fun g us invades through the cortex until it reaches the vascular tissue . Durin g the early sta g es of disease development, the fungu~ i$ ~estricted mainly to the xy lem and x ylem parench y ­ ma. In later sta g es, before th e host dies, the fun g us moves out of the xy lem and invades surroundin g tissues. After death of the host plant, abundant microsclerotia are pro­ duced throughout the infected tissue and as the plant decays, these microsclerotia become part of the soil complex . Under usual conditions of soil moisture and temperature microscle­ rotia seem to stay viable for several years. Accordin g to
Garrett's ( 6 , p. 30) e cological g roupin g of soil fungi V. albo-atrum can be classified as a root-inhabiting fun g us without the ability to g row saproph y ticall y in soil.
The fun g al structures usuall y called microsclerotia, sometimes called psudosclerotia ( 32 , p. 9 ), are responsible for survival of the or g anism in the absence of host plants.
Microsclerotia are black, multicellular bodies found in dead plants in nature, and are readily produced in culture media.

2
They are produced by the rounding up of adjoining cells at intervals along hyphae , followed by thickening and darkening of their cell walls .
Several investigators (3 , 11 , 25 , 33) have demonstrated the influence of soil moisture and temperature on disease severity , but the influence of these factors have not been studied with respect to microsclerotia . The fact that microsclerotia are the principal agents of dissemination and infection warranted a thorough study of the influence of environmental factors on their survival . Experiments re­ ported in this thesis were designed to determine the effect of soil moisture and temperature on survival of the micro ­ sclerotia of V . albo atrum .

3
Reinke and Berthold (31) first described Verticillium albo-atrum R .
B . as causal agent of a vascular disease in potatoes . Since then V. albo-atrum has been reported to incite disease in many species of plants (2 9 , 33, p. 261).
In 1 9 13 Klebahn (1 6 ) described a Verticillium isolated from dahlia which he called Verticillium dahliae Klebahn . He stated that the fungus isolated from potatoes by Reinke and
Berthold produced only dark mycelium whereas the one iso­ lated from dahlia produced microsclerotia in culture .
Rudolph (3 3 , p . 2 4 7-252) in 1931 concluded that the potato fun g us produced true microsclerotia even though there was no clear description by the ori g inal describers of the species.
Van Den Ende ( 4 1) concluded from his studies that Verti­ cillium albo atrum cannot be distin g uished from Verticillium dahliae on a morphological basis and hence the former name, having priority, is correct . There is a g reat deal of con­ fusion in the literature on taxonomy and nomenclature of
Verticillium species . The fungus used in studies reported in this thesis produces abundant microsclerotia , but will be called Verticillium albo-atrum R .
B. in accordance with the views of Rudolph (3 3 , p. 151) and Van Den Ende ( 4 1).
The term microsclerotia has been extensively used in the literature . In this thesis it is considered in accor­ dance with the definition of Snell and Dick ( 3 5 , p . 96) as

4 na small clump of dark-colored , more or less thick-walled cells , each of which is viable , produced in the medium of cultures and rarely in the xylem of host plants by Verti­ cillium spp . and possibly other Fungi Imperfect!."
The literature on Verticillium wilt diseases is volu­ minous , but there is relatively little wor k reported on the microsclerotia of Verticillium . It is lo g ical to believe that the importance of microsclerotia as the restin g bodies of Verticillium was not realized until 1 9 13 when Klebahn
( 1 6 ) first isolated Verticillium from dahlia*
Presley ( 3 0) reported that under unfavorable condi­ tions , the fun g us may overwinter as microsclerotia . Nelson
(25, p . 1 6 7) who conducted the ori g inal studies on Verti­ cillium wilt of peppermint stated that Verticillium is cap­ able of livin g in the soil for at least 12 years in the ab­ sence of cultivated plants . This ability he attributed to the saprophytic nature of the fun g us . Wilhelm ( 6 ) found that Verticillium surviv e d in laboratory culture for 1 3 years and for 1 4 years in the field .
Luck ( 1 8 ) de onstrated that Verticillium would not col­ oni z e muc k soil . He could not recover the fun g us after a period of 3 weeks when soil was arti f icially inoculated with a heavy suspension of mycelium and spores free from micro ­ sclerotia . Isaac (10) workin g with Verticillium albo atrum and V . dahliae noticed that both fun g i were incapable of spreadin g in the soil as saproph y tes . Wilhelm ( 4 5 , 47 ) did

5 extensive work to study the ability of Verticillium to grow through the soil and colonize pieces of tomato ste placed at a distance from the inoculum . He concluded that Verti­ cillium in no instance gr ew through unsterile soil nor did it colonize the tomato stems . However , Wilhelm obtained ra ­ pid g rowth of Verticillium throu g h sterile soil . Wilhelm
( 46 , p . 181) also stated that "since Verticillium persisted for 13 years in culture , during at least 12 of which survi­ val was by eans of microsclerotia in the dry state , per­ sistence for a similar period , 1 4 years in field soil with no hosts present , is thought also to reflect the longevity of the resting structure . " Thus it is probable that the fungus survives in th e soil only as microsclerotia .
The number of microsclerotia present in a unit volume of soil has been called the inoculum potential ( 44 ) .
Wilhelm ( 44 ) working with tomato plants and Tolmsoff ( 3 9 , p . 112) with potato plants found that the severity of wilt was directly proportional to the inoculum potential . Tolm­ soff ( 39 , p. 11 3 ) also showed a correlation between the num­ b er of actual infections on roots and disease severity .
The thermal death point of Verticillium microsclerotia was determined to be 1 0 minutes at 50 ° C or 40 minutes at
4 7 ° C (2 4 ) . Sli g htl y higher temperatures were reported by
Luck (1 9 , p . 70-72) . Microsclerotia exposed at i9 ° to 50 ° C in a dry atmosphere survived for 6 months while conidia died in 3 days (2 4 ) . Microsclerotia of a different isolate

6 however , survived 2! years at a temperature of 4 0° c .
Studies conducted at room temperature showed that micro ­ sclerotia survived desiccation over 17 months ( 19 , p . 75) .
The writer observed similar survival results when micro ­ sclerotia were artificially mixed with air-dry soil and kept at room temperature over a period of one year . The number of survivin g microsclerotia declined g radually during this time .
Ability to survive desiccation is also a characteristic of sclerotia of other fun g i. T he sclerotia of many fungi , however , have little in common with the microsclerotia of
Verticillium . True sclerotia are formed from a mass of h y phae , whereas the microsclerotia are formed intercalarily from sin g le h y pha and in this respect resemble chlamydo ­ spores rather than sclerotia. However , all of these struc ­ tures are restin g bodies , resistant to adverse environmental factors to a certain de g ree , and g enerall y function to carry the fun g us throu g h periods of adverse conditions .
Sclerotia of Corticium va g um survived 237 da y s of stora g e in a corked tube ( 2 8 ) . On the other hand , sclerotia of Ph y matotrichum omnivorum were k illed when air dried for l i hours in the laboratory at room temperature (1 4 , p . 8 2 4 ) .
Nisikado and Hirata ( 2 6 , p. 546 ) reported that sclerotia of
Sclerotium or y zae remained viable in air dry condition for periods of 3 years at 2 0 ° , 1 year at 25• C and 4 months at
35 ° C . Sclerotia of these fun g i show variation in

7 resistance to dryin g . A similar variation in resistance to chan g es in soil moisture and temperature is a characteristic of the sclerotia of several fun g i .
King and Eaton (15) reported that sclerotia of
Phymatotrichum omnivorum k ept in air-dr y soil , as well as those k ept in soil containin g 1 0 % moisture showed g radual decline in lon g evity after a period of 2 months with onl y a few surviving at the end of a year . Best survival was obtained at 25$ and 2 8 % soil moisture levels . In the above tests percenta g e moisture was calculated on the basis of field capacity which was taken as 1 00 % . T aubenhaus and others (37) have reported that floodin g for 12 0 days failed to eliminate the fun g us from the soil , mainly due to the presence of sclerotia in the field . Taubenhaus and Ezekiel
( 38 ) found that sclerotia of Ph y matotrichum omnivorum at room temperature could survive up to 5 or more years , when k ept in moderately moist soil .
Sclerotia of Sclerotium ory z ae remained viable for
6! months on air dry rice soil in the shade , while buried in moist soil the y survived
4! months , and when submer g ed in water the survival time was 1 0! months (2 7 ) . But Tullis and Cralle y ( 40 ) found that the sclerotia survived for 6 years in uncultivated rice soil . As neither of these in­ vesti g ators recorded the temperature it would be hard to e x plain t hese contradictin g results . Higgins ( 8 ) su g gested

8 that it is not likely that S. oryzae sclerotia would last long under field conditions because of their suseptibility to freezing when wet .
Sclerotia of Rhizoctonia solani were found to be viable after immersion in tap water for 571 days and also after bein g in a dry corked tube for 237 days (2 8) . Wardlaw ( 43 ) has shown that the banana wilt pathogen Fusarium oxysporum cubense , which produces chlamydospores , can be controlled by prolonged flooding .
Verticillium was found to be "active in the field" in all moisture levels above the wilting point ( 34 , p . 142) .
In cotton , Le y en d ec k er (17) found an effective ran g e of in­ fection from 25% to 85% of field capacity . Conflicting re­ sults w ere obtained by Isaac ( 9) , where increased soil moisture decreased the disease incidence . Later Isaac (11) pointed out that an increase in the amount of moisture favors the fungus .
These results where the survival rate of the fungus with varying moisture levels is measured by the incidence of disease in plants may not be accurate due to th e influ­ ence of soil moisture on host plants themselves . Nelson
(2 5 ), however , su gg ested that soil moisture could influ­ ence survival of Verticillium. The differences in severity of dis ease at various levels of moisture might be due to a difference in the inoculum levels .

9
There is considerable discrepancy in reports of vari­ ous investi g ators on the effect of temperature on survival of fungus resting bodies in the soil . Variation is found not only among different fungi , but also is evident in the results of workers studyin g a single fun g us , probably be­ cause of differences in techniques and fungus strains .
King et al (1 4 , p . 839) found that sclerotia of Phyma ­ totrichum omnivorum could be killed in 2 to 4 minutes when placed in soil k ept at 43 ° C. But it took 15 minutes to kill the same sclerotia immersed in hot water at 46 ° c.
Park and Bertus ( 28) reported that sclerotia of Rhizoctonia solani were viable after exposure to the sun for 28 4 hours .
Walker ( 4 2 , p . 700) found that with Sclerotium cepivorum infection occured readily at soil temperatures b etween 1 4 ° and 1 8 ° C. Between 22 ° and 2 4 ° C infection was reduced markedly and there was no infection above 2 6 ° c.
Interestin g results were obtained with Sclerotium rolfsii by Epps et al ( 4 , p . 25 4 ) . T hey found that maximum survival of the fungus occured between 30° and 35 ° C . This survival gr aduall y decreased with a reduction in the soil temperature and below 15° C the nactivity'
1 was completely stopped .
Helminthosporium sativum and Fusarium lini were found to survive in the soil at temperatures up to 37 ° C, but were almost eliminated in 6 days at 4 5° to 50 ° c

10
{2 , p . 1015) . It has been reported that chlamydospores of
Ustilago nuda would survive temperatures of 28° to 38 ° F for 12 to 13 years {36) . Merek and Fergus { 22) observed that ascospores of the oak wilt fungus survived for 3 days at 37 ° C while at 24° they were viable up to 81 days . But wetting the spores at 2 4 ° decreased their longevity by half . It has been noticed by the writer that spores of any common soil fungi can survive a temperature of 4 0 ° C for a considerable time .
There have been many studies carried out to determine the minimum , maximum and optimum temperature requirements for growth of Verticillium { 19 , 4 5) . Most of th e se studies were conducted with artificial culture media . As pointed out by Rudolph (3 3 , p . 2 30 ) several writers have agreed that Verticillium wilt is mainly a disease confined to cool areas . However , the validity of this opinion seems debat ­ able when one considers that Verticillium wilt is a serious disease of cotton in the irrigated , arid Southwestern United
States . Otherwise , most of the work has been directed towards the effect of soil and air temperatures on symptom e x pression .
Even thou g h the fungus in the soil is affected directly by temperature , there is always the unknown factor of in ­ direct effect of soil temperature on the host physiology .
Again , different hosts may react differently at the same

11 soil temperature and inoculum level . To overcome these problems in studyin g the influence of soil temperature a direct method of measuring the survival rate of the fun g us seems desirable .
Ludbrook (2 0 , p. 152) reported a difference in sus ­ ceptibilit y between hosts at higher te peratures . He stated that the highest incidence of wilt in tomato inocu ­ lated with Verticillium albo -a trum occurred at a soil tem­ perature of 26° compared to 30° C · in eggplant inoculated with the same isolate . The air temperature in each case averaged 20° C . Ludbrook also studied the influence of soil temperature on several Verticillium isolates and con ­ cluded that (20 , p . 151) ''The most important environal factor influencin g th disease appears to be temperature ."
All the isolates he studied produced severe disease betwe e n
1 8 ° and 22° C . Above 2 4 ° C the disease severity decreased rapidly while there were no symptoms at 28° to 30° c .
He also reported a difference in the severity of symptoms pro­ duced by microsclerotial and non-microsclerotial isolates of Verticillium . Although not stated by Ludbrook , this ay have been due to a difference in resistance of micro ­ sclerotia to te perature or to a difference in inoculum potential of the two isolates.
Alexander (1, p . 62) studied the influence of soil temperature on wilt severity in tomatoes . He stated that

12 there was severe wilt fro 12• to 2 4 ° C while plaots made noraal growth showioa only ild yaptoms at 2 8 ° C . Very similar results ar reported by McKeen (21, p . 11 5 ). Be fouod that a miorosclerotial isolate of Verticillium pro­ duced aaximua disease in tomato plants t a soil t apera­ ture of 2 4 ° c.
At 3 0 ° the plant were either healthy or ahowed only mild symptoms . ScbDeider (3 4 , p. 1 4 2) studied the effect of soil temperature on develop at of Verti­ cilliua wilt in auayule and found that the fungus became iDactive between 80 ° aDd 85 ° P .
The investigations r viewed s o f ar have be D restric­ ted to the influence of soil temperature and aoistur on disease incidence . Bdgiogtoo aDd Walker (3) receotly com­ pared the effect o f temp rature on growth of two Verti­ cilliua isolates aod evaluated separately the iDflueoce of soil aDd air temperatures oo disease development . The
"dauermycelial" isolates of Vertieillium albo-atrua grew well at 22• C. They erew slowly at 2 s • while at 32° there was no visible growth . The optimua temperature for the pseudosclerotial isolates was 2 4 ° C. Growth was reduced only slightly at 2 8 ° and there was only littl growth at
32° C . The ••daueraycelial'' isolates produced severe symp­ to s on to11ato plaot at combinati o ns of 2 0 ° t o 2 4 ° soil temperature with 1 6 ° to 2 4 ° air teaperature. But at a soil te perature of 2 8 ° witb air teaperaturas of 1 a • to 2 0 •

13
C there was only slight disease , while no symptoms appeared
' when both air and soil temperatures were 28° C . In con ­ trast the pseudosclerotial isolates induced severe disease in both tomato and cotton at all the above combinations of air and soil temperature . From this study the investiga ­ tors concluded that both factors had significant effect on disease development, but soil temperature was the more im ­ portant of the two .
A lack of information of the influence of moisture and temperature on survival of Verticillium microsclerotia in soil is evident . Almost all investigations reported in the literature were conducted either to study the effect of soil moisture and temperature on th9 incidence of disease caused by Verticilliu , or on the ability of the fungus to survive in soil as a saprophyte . Attempts have been made by different investigators to study the longevity of micro­ sclerotia on culture media ~ Microsclerotia are of consid­ er ble importance in the survival of Verticillium as well as in disease incidence and severity . For this reason a study of the effect of soil moisture and temperature on their survival was carried out .

1 4
METHODS AND MATERIALS
CULTURE
An isolate of Verticillium obtained from a severely wilted peppermint plant from a mint field near Jefferson,
Oregon was used throughout this study. This isolate is designated as No. 91 in the collection of Dr. C. E. Horner at Oregon State College. On potato-dextrose agar it pro­ duced microsclerotia in great numbers and its pathogenicity to peppermint was proved. As mentioned earlier this iso­ late is regarded in this thesis as Verticillium albo-atrum.
MEDIA
In the course of this investigation potato-dextrose agar containing 100 ppm. streptomycin was used to make iso­ lations from peppermint plants as well as for growing inocu­ lum. The medium used for making dilution plates will be described under the section on soil dilutions.
PREPARATION OF INOCULUM
The method used was a modification of that described by Luck (19, p. 61). Verticillium was grown on water­ permeable cellophane in 10 X 12 inch plastic containers.
These co nt ainers were sterilized in two steps. First, they were thoroughly swabbed with 50% commercial Clorox
(5.25% sodium hyp0clorite) and allowed to stand for 2 4 hours with a ljttle Clorox ~n the bottom. After this

1 5 period the Clorox was washed out w ell and the containers were rinsed with 9 5% ethyl alcohol. Sheets of cellophane were out to fit the botto of the containers and sterilized in the autoclave. Three hundred ml. of melted streptomy­ cin-potato-dextrose agar was poured into each container and allowed to solidify. Cellophane sheets were dipped in sterile water and laid on top of the semi-solid agar.
Dipping in water expanded the cellophane resulting in a smooth surface when laid on agar. Spores of Verticillium suspended in sterile water were atomized onto the cello­ phane surface. The containers were then incubated for 1 0 to 1 4 days in the dark at room temperature.
Within two weeks the fungus produced an abundance of microsclerotia. These were scraped off the cellophane sheet with a razor blade and blended in a Waring blender for 2 minutes with an equal amount of water. The result­ ing slurry of microsclerotia and water was mixed with well­ screened and air-dried soil. When the inoculated soil be­ came dry the small clumps were broken and the soil was again thoroughly mixed, then allowed to dry at room tem­ perature for 5 to 7 days. Drying eliminated most of the viable conidia as well as pieces of mycelium. Dilution plates were poured from the dried inoculum to determine the approximate number of microsclerotia per cubic centimeter of soil. The microsclerotial population was found to

16 remain quite constant for several months when kept at
C .
However , for each experiment a fresh batch of inoculum was used .
In each experiment a calculated amount of stock inocu­ lum was mixed with air dried and well screened field soil to bring the experimental soil to the desired inoculum level . Mixing was dooe iD a cement mixer to obtain an even distribution of microsclerotia in the soil .
SOIL
A Chehalis sandy loam soil was used iD all experi ­ ments . This soil was obtained from the Botany and Plant
Pathology Farm located near Corvallis , Oregon . No attempt was made to sterilize the soil , however , tests showed that it contained no detectable amount of Verticillium . Before incorporating the inoculum this soil was well screened to remove lumps and large particles of plant material .
SOIL MOISTURE
Th various levels of soil moisture used were : air dry; 25 , 50 and 75% field capacity; field capacity , aud flooded . Field capacity was determined by flooding the soil in a can with p rforated bottom and allowing the excess water to drain off by gravity . This was done in a cold room at 5° C so that there would be minimum water loss from the soil by evaporation . The amount

17 of water left in the soil after 5 days drainage was deter­ mined on an oven dry basis and this was taken as field ca­ pacity, which was found to be 22% by weight .
The fie ld soil was w ell screened and allowed to dry in air for several days. San1ples wer taken and the amount of moisture in this dried soil was obta ined on an oven dry basis. Then batches of soil were brought to the required mois ture levels by adding a calculated amount of water to a known amount of soil. Soil was weighed and fed into a ce ent mixer nd the required amount of water was added in the form of a fine spray using a spray gun . By this method thorough mixing nd even distribution of moisture were ob­ tained.
A calculated amount of stock inoculum was thoroughly mixed with each batch of soil in the cement mixer . This inoculated soil was measured out into containers . Wide­ mouthed gallon jars containing 7 pounds were used to keep soil in green-house temperature tanks while plastic boxes containing 300 grams were used to keep soil in constant temperature cabinets. These amounts left adequate air space above the soil level . The screw-type lids on the glass jars and a band of tape around the plastic boxes helped minimize loss of moisture by evaporation . Consi­ derable water loss occurred in containers kept at tempera­ tures above 25° C while there was only slight loss at 15•

aod 2 0 °. There was no measureable loss of water from con­ tainers at s• and 1 0 •. A record of the weights of these containers was kept and water was added once a week to com­ pensate for loss by evaporation. After each addition of water the soil was thoroughly mixed to insure an even dis­ tribution of moisture.
-
SOIL TEMPERATURE CONTROL
E:x:perim .
ents were conducted in both soil temperature tanks and constant temperature cabinets. The soil tempera­ ture tanks were supplied with heating coils and refrigera­ tion units. Water temperature was thermostatically con­ trolled. It was possible to maintain the temperature in the tanks to plus or minus 1• c.
Even this fluctuation was minimized by checking the temperature every day and making necessary adjustments whenever needed. Four units of the tanks were used. Each unit held 15 jars of soil. The tem­ peratures were adjusted to 10•, 20°, 30° and 40° C and checked twice a day for a period of 7 days before the ex­ periment was started.
The constant temperature cabinets m aintained temper­ atures at s•, 1 0 •, 1 5 °, 2 0 •, 25° and 3 0 • c. Fifteen plas­ tic boxes with s o il were placed in each cabinet.
SAMPLING
Three replications of each of 5 moisture levels were
1 8

19 available for aualysis . Sampling was conducted by thoroughly aixing the soil mass and then r moving a 5 gram sample . S amples were k pt in a cold room at
C for 2 days before soil dilution plates were poured . Being a com­ parative study , no attempt was made to compensate for the weight of water in soil samples with different oisture levels . Nevertheless , care was taken to be consist e nt in sampling methods .
Samples from the soil temperature tanks were taken once every three months . For sake of convenience the first half of the treatments were sampled on one day and the second half were sampled on another day .
Soil from the temperature cabinets was sampled once a month . Here also for convenience the first half was sampled on one day and the second half another day .
One of the major problems in this study was to de­ velop a technique to determine the number of viable micro ­ sclerotia in the soil . The use of test plants was not considered feasible since it does not give a clear quan­ titative measure of the surviving microsclerotia . Use of plants also r quires a large quantity of experimental soil and a long p riod to obtain results . It was decided to develop a laboratory method to determine the number of microsclerotia in a given sample of soil . Agar dilution

- - - - - - - - - - - -
20 plate methods are commonly used for determining populations of soil fungi, but in preliminary studies it was found that large numbers of soil fungi masked the presence of Verti­ cillium on conventional agar dilution plates. At this stage a separation of microsclerotia from the spores of other soil fungi by specific gravity methods was tried.
This was unsuccessful since the density range of micro­ sclerotia and many otber fungus spores was similar. A suppression of unwanted fungi on dilution plates was also tried. By the -.d~ition of oxgall reduction in radial growth of m-DY fungi was ob ta iDed. Addition of strepto­ myciD reduced the number of actinomycete and other bac­ terial colonies. Most chemicals incorporated with the media either had DO effect or inhibited the germination of
Verticillium along with other fungi.
It was noticed, however, that ethyl alcohol added to water agar resulted in readily distiDguishable colonies of Verticillium while other fuDgi grew sparsely or not at all. After about teD days colonies of Verticillium pro­ duced microsclerotia and turned black and were easily rec­ ognized from other dark colored fungi on dilution plates.
Verticillium colonies stood out sharply in contrast to other fungi when viewed against a white background. The use of this medium to determine the microsclerotial popu­ lation of Verticillium iD artificially inoculated soil was

21 described (23) . Unlike potato-dextrose agar , plates poured with alcohol agar medium can be held for many days without any reduction in the accuracy of determination .
Throughout the course of this study alcohol agar medium was used to assay the microsclerotial population by soil dilution . The medium cont ined per liter 7 . 5 grams agar , 0 . 5 ml . 20% streptomycin nitrate and 5 ml . absolute ethyl alcohol . It ~as made up in the usual manner, but the alcohol was added just before the plates were poured in order to prevent loss by ev poration .
All dilutions were made on a volume to volume basis in sterile distilled water. Five grams of soil were mixed with 20 0 ml . of water and shaken well for one minute .
Dilutions were then made by pipetting 10 ml . into 90 ml . of water . The final dilution was made into 9 0 ml . of alcohol agar medium and poured into 10 petri plates . Streptomycin ­ water agar in 250 ml . Erlen yer flasks was held at 44• C in a water bath . One half ml . of absolute ethyl alcohol was added to these flasks before diluted soil samples were added . The plates were incubated in the dark at room tem ­ perature for ten days . At the end of this period Verti ­ cillium colonies were counted and recorded . The total num ­ ber of Verticillium colonies present on 10 dilution plates was multiplied by the dilution factor to obtain the actual count of viable microsclerotia in a sample .

22
The two dilutions used in pouring plates were
1 : 10 , 000 and 1 : 100 , 000 . However , towards the later stages of the experiment 1 : 1000 dilutions were used since there were no Verticillium colonies in the higher dilutions .
Lower dilutions than 1 : 1000 were not used because of di­ minished accuracy in counts due to a variety of fungal colonies present in large numbers .
In the course of this investigation certain disadvan ­ tages of the dilution technique were noticed . Variation in counts due to an uneven settling of soil particles in dilution flasks was one of them . Attempts were made to minimize this by adding a substance that kept the soil particles an.d microsclerotia in an even suspension . The use of 0 . 5% high viscosity carboxy methyl cellulose (CMC ;
4 8, p . 3) in the initial dilution helped to suspend the particles uniformly .
This method was further standardized by shaking the initial dilution for one minute and allowing the flask t o stand for 10 seconds before pipetting out the 1 0 ml . por ­ tion . These aliquots were pipetted from a constant level in the flask .
ASSAY BY INDICATOR PLANTS
In addition to assaying microsclerotial population by soil dilutions , the experimental soil was assayed to deter ­ mine infection and symptoms of wilt produced in indicator

23 plants . At the end of the first two experiments rooted peppermint cuttings were planted in the experimental soil .
Whenever there was not enough soil in a replication to plant a cutting all three were mixed together . Cuttings were grown in this soil for 2 months which is known to be adequate time for plants to show wilt symptoms under usual conditions of growth . At the end of this period sections from base , middle and top of the main stem were taken for isolation tests . These sections were surface sterilized by dipping in a 50' solution of commercial Clorox and placed on a clean paper towel , then three smaller sections , about o .
s em . in length , were cut from the middle of the larger pieces with a flamed razor blade . Using flamed tweezers these pieces were transferred to streptomycin ­ potato dextrose agar plates and incubated for 3 to 4 days at room temperature . The plates were then examined for the presence of Verticillium .

24
It is evident from the literature that Verticillium survives for several years in soil as microsclerotia and that disease severity is directly proportional to the num ­ ber of microsclerotia in soil . Microsclerotia are princi­ pal agents of dissemination and survival of Verticillium , and their concentration in soil determines disease sever ­ ity ; therefore a knowledge of the effects of soil te per­ ature and moisture on the survival of microsclerotia is basic to a better understanding of the biology of this fungus . Experiments were conducted to determine the in­ fluence of soil moisture and temperature on survival of
Verticillium microsclerotia .
E
T
T
In July 1958 , a test was started to determine the ef­ feet of soil moisture and temperature on Verticillium mi­ crosclerotia . Dry field soil was infested with Verticil­ lium microsclerotia at the rate of 2 . 3 million per cubic centimeter then adjusted to moisture levels of air-dry ,
25 , 50 , 75% field capacity and flooded. Three replica ­ tions of each moisture level were placed in soil temper­ ature tanks at each of four temperatures (10° , 20° , 30° and 4 0° C) . First samples were taken on July 26 , 1958 and successive samples were taken at three month intervals .

25
Sampling was continued until the end of January, 1959. At that time all remaining soil was planted to rooted pepper­ mint cuttings. a) Results as determined by soil dilution:
Microsclerotia survived at temperatures of 10° and 20°
C in soil at all moisture levels except flooded for over 6 months (Table 1 and 2). Viable microsclerotia were not obtained from flooded soil after the first sampling. How­ ever, this does not necessarily mean that all microscle­ rotia were dead because the dilution technique used was not sensitive enough to detect viable microsclerotia when present in very low numbers.
At 30° C there was a great reduction of microscle­ rotia in all moisture levels within a period of three months and after six months it was not possible to recover viable microsclerotia on dilution plates.
Soil temperature of 40° C was found to reduce the population within 3 weeks to a level where it could no longer be detected by the dilution technique. b) Results as determined by plant assay:
The results of the isolations made from peppermint plant (Table 3) were found to correspond to the results obtained by soil dilution. Of 15 plants grown in soil that had been held at 10° c,
13 showed symptoms and 14

TABLE 1 . Survival of an initial 2 . 3 million Verticillium microsclerotia per cc over a six month period in soil held at 5 levels of moisture and 4 levels of temper­ ature .
Number of viable microsclerotia detected ( Millions per cc)
10° c
20° c
30° c
40 ° c
Moisture levels 2* 1 4 26 2 1 4 26 2 l 'X 26 2 1 4 26
Air-dry
25% F .
2 . 4 2 . 2 0 . 5 1 . 1 0 . 5 0 . 01 0 . 5 o .
o
0 . 0 o .
o
0 . 0 0 . 0 c .
2 . 4 1 . 8 0 . 4 2 . 3 0 . 6 0 . 05 0 . 7 0 . 01 0 . 0 0 . 0 o .
o o .
o
50% F .
75% F .
Flooded c .
5 . 2 5 . 7 1 . 0 4 . 9 1 . 2 0 . 08 2 . 6 0 . 02 o .
o o .
o
0 . 0 o .
o c .
5 . 9 4 • '~ 0 . 2 13 . 8 1 . 0 0 . 01 0 . 5 0 . 01 o .
o
0.0 o .
o
0 . 0
1 . 3 0 . 0 0 . 0 0 . 03 o .
o
0 . 0 0 . 05 0.0 0 . 0 o .
o o .
o o .
o
* Number in weeks after the test was started.
TABLE 2. Percentage reduction of Verticillium microsclerotia in soil after a six month period at 5 levels of moisture and 4 levels of temperature.
Moisture levels
10° c
20° c 30° c 40° c
Ai r-dry
25%
50%
F . C .
F . C .
75% F . C.
Flooded
79 . 1
83 . 3
80 . 7
96 . 6
100 . 0
99 . 0
97 . 8
98 . 3
99 . 9
100 . 0
100 . 0
100 . 0
100 . 0
100 . 0
100 . 0
100 . 0
100 . 0
100 . 0
100 . 0
100 . 0 l'l) en

TABLE 3 .
Moisture level
Isolation results from the temperature tank experiment showing effect of mois­ ture and temperature on the population of Verticillium microsclerotia in soil as assayed by indicator plants . Soil samples were held for six months at in­ dicated moisture and temperature.before assayed .
10° c
20° c
30° c
40° c
Plants Plants showing sy ptoms
Plants infected showing symptoms
Plants Plants Plants infected showing symptoms infected
Plants Plants showing symptoms infected
Air-dry 3
25$ F . c .
2
50$ F . c .
3
75$ F . c .
3
Flooded 0
3
3
3
3
0
1
2
2
3
0
2
3
3
3
0
0
0
0
1
0
0
0
2
1
0
0
0
0
0
0
0
0
0
0
0

28
Figure 1. Coaparisoa of growth of pepperaiDt plaDta after two aoatha iD Vert1c1111ua-1Dfeat•d aoila at three levels of •oiature. Tba aoil• had bee• iacubated for six aoutha at 10• C prior to pl&DtiDg.

29
Figure 2. Comparison of growth of pepp raint plants after two aonths in Verticilllua-infested soils at three levels of aoisture. The soils had been incubated for six aonths at 20• C prior to planting.
/

30
Figure 3. Comparison of growth of pepperaiut plants -fter two months in Verticillium-infested soils at three le¥els of moisture. The soils had been incubated for six months at 30° C prior to planting.

31
Figure 4. Comparison of growth of peppermint plant• after two months in Verticilliua-infested soila at thr•e levels of moisture. The soils had been inc~bated for six modths at 40° C prior to plaeting.

32 were infected . This included 2 of 3 plants grown in soil that had been flooded . Of 15 plants grown in soil that had been held at 20° c , 11 were infected , but only 8 of the 11 developed symptoms . Plants grown in soil that had been flooded were all healthy . Fifteen plants were grown in soil that had been held at 30° c , but only 3 plants became infected . Of the three infected plants one plant grown in soil that was held at 75~ field capacity showed mild symp ­ toms . None of the plants grown in soil held at 40° C were infected . Figures 1 to 4 show a comparison of plants grown in soil held at each level of temperature and mois ­ ture .
TEMPERATURE CABINET EXPERIMENTS
Preliminary experiment
A batch of the same infested soil used for the tem ­ perature tank experiment was adjusted to moisture levels of air-dry , 25 , 50 and 75% field capacity and flooded , then held in plastic boxes at 5° , 10° , 15° , 20° , 25° , and
30° c .
The five moisture treatments were replicated three
.
times at each temperature . First samples were taken on
July 19 , 1958 and successive samples were taken once a month until the end of February , 1959 . At this time rooted peppermint cuttings were planted in the experimental soil .

33
TABLE 4. Effect of different levels of soil moisture at
5° C on survival of Verticillium microsclerotia.
No. of viable Microsclerotia (Millions per cc)
Moisture levels Jul~ Auf!. Sept. Oct. Nov. Dec. Jan. Feb.
Air-dry 2.5 1.0 3.6 1.8 3.0 2.1 1.5 0.3
25% F.
50% F.
75% F. c. c. c.
1.7
2.8
8 . 4
Flooded 1.8
1.7
9.5
14.4 o.s
2.3
1.1
9
0
.1
.2
0.9
3.0
8 .6
0.1
3.7
4 .6
5.9
0 .02
1.4
4
8
0
.
.
.
9
0
01
1.1
4 . 0
5.8
0 .001
0 .3
1.3
1.0
0.001
TABLE 5. Effect of different levels of soil moisture at
10° C on survival of Verticillium microsclerotia.
No. of viable Microsclerotia (Millions per cc)
Moisture levels July Aug. Sept. Oct. Nov. Dec. Jan. Feb.
Air-dry 1.0 2.4 3.3 2.7 1.9 1.4 2.0 0 . o
25% F.
50% F.
75% F. c. c. c.
0
8
.
.
8
0
7.8
0
3.6
8
.
.
9
8
1.3
2.1
6.5
0 . 8
1.4
3.6
0
3.6
7
.1
.7
1.6
6 .3
5.9
Flooded 1.6 0 . 6 0 .1 0 . 02 0.003 0 . 001
2.2
2.9
3.
o
4
.o
0 . 7
1.2
1.0 o .o
TABLE 6. Effect of different levels of soil moisture at
15° C on survival of Verticillium microsclerotia.
No. of viable Microsclerotia (Millions per cc)
Moisture levels July Aug. Sept. Oct. Nov. Dec. Jan. Feb.
Air-dry
25% F.
50$ F.
75% F.
1.1 0 .1 0 .0
8 0.001 0.001 o .
o o .
o o .
o c. 0 .7 0 . 04 0 . 003 0 . 001 o .
o o .
o o .
o o .
o c.
0 .2 0 .3 0 .0
4 0 .01 0 .02 0.02 0.01 0 .01 c.
0 .3
Flooded 0 . 01
0.03 0 .001 0.02 0 .01 0 .02 0 .01 0.01 o.o o .
o o.o o .o o .
o o.o o .o

34
TABLE 7. Effect of different levels of soil moisture at
20° C on survival of Verticillium microsclerotia.
No. of viable Uicrosclerotia (Millions per cc)
Moisture levels July Aug. Sept. Oct. Nov. Dec. Jan. Feb.
Air-dry 1.6 0 . 9 0 .3 0 .1 0.1 0 .1 0 . 0 0 . 001
25% F.
50% F.
75% F. c. c. c.
0
4
4
.1
.
.
0
8
0 . 4
2.0
2.4
Flooded 0.2 0 .1
0 . 3
2.3
2.3 o .
0
1.0
0 o o .
.1
. 8
0 . 04
1.2
0.8 o o .
o
1.0
0 o o .
.
. o
4
0
0
0 o o .
. 001
.1
.1 o
0
0 o o .
.
. o
3
.1 o
TABLE 8. Effect of different levels of soil moisture at
25° C on survival of Verticillium microsclerotia.
No. of viable Microsclerotia (Millions per ec)
Moisture levels July Aug. Sept. Oct. Nov. Dec. Jan. Feb.
Air-dry 0.7 0.3 0 .1 0.03 0 . 01 0 . 0
25% F.
50% F.
75% F. c. 0.03 0.03 0 . 02 0 . 001 0 . 001 o .
o .
o o.o o o .
o o .
o c. 1.9 1.0 0.6 0 .2 0 .1 0 . 04 0 . 02 0 . 02 c.
9 . 6 1.1 0.4 0 .1 0 .1 0 .1 0 .01 0 . 02
Flooded 0.2 0 . 002 o .
o o .
o o .
o o .
o o .
o o.o
TABLE 9 . Effect of different levels of soil moisture at
30° C on survival of Verticillium microsclerotia.
No. of viable M icr osclerotia (Millions per cc)
Moisture levels July Aug. Sept. Oct. Nov. Dec. Jan. Feb.
Air-dry
25% F.
50% F . c.
1.1
0 . 7
0.1
0 . 04
0 . 03 0 . 001 0 . 001
0 .0
03 0 .001 o .
o .
o o o .
o o o
.
o
.o o o
.
.
o o c.
0 .2 0 .3 0 .0
4 0 . 01 0 . 02 0 . 02 0 . 01 0.01
75% F. c. 0 . 3
Flooded 0 . 01
0 . 03 0 . 001 0 .02 0 . 01 0 . 02 0 . 01 0 . 01 o .
o o .
o o .
o o .
o o .
o o .
o o .
o

TABLE 10 . Percentage reduction of Verticillium microsclerotia after a six month period in soil held at various Ieveis of moisture and temperature .
Moisture level 5° c
10° c
15° c
20° c
25° c
30° c
Air-dry
25% F . c .
50% F . c.
75% F . c .
Flooded
88 . 0
82 . 3
53 . 5
88 . 0
99 . 9
20 . 0
12 . 5
85 . 0
87 . 1
100
88 . 2
99 . 9
60 . 8
91 . 9
100
99 . 9
100
92 . 5
97 . 9
100
100
100
98 . 9
99 . 7
100
100
100
95 . 0
96 . 6
100

36 a) Results as determined by soil dilution:
Microsclerotia in soil held at 5° C survived for over
7 months at all moisture levels (Table 4) . Maximum survi­ val was obtained in soil at 50% field capacity (Table 10) .
Similar counts were obtained from soils held at 10° ,
15° and 20° C (Tables 5, 6 and 7) . Verticillium colonies were not detected , however, after being in flooded soil for 5 months. There was also reduction in survival as the temperature increased from 10° to 20° c.
Microsclerotia were killed rapidly at 25° and 30° C
(Tables 8 and 9) . No colonies were detected in air-dry and 25% field capacity soil treatments after 4 months , while in flooded soil Verticillium was not recovered after one month . In soil held at 25° and 30° C detectable sur­ vival was best at 50 and 75% field capacity . b) Results as determined by plant assay:
After soil dilution assays were completed , rooted peppermint cuttings were planted in the remaining soil .
Because only a limited amount of soil was available , the
3 replications were consolidated and a single cutting planted in each . After 2 months plants were examined for symptoms and then stem sections removed for isolation .
The isolation results are presented in Table 11 . All
5 plants in soil held at 5° C were infected and 4 of the 5 showed symptoms . In soil held at 10° and 15° C all plants

37 ex cept those in the flooded soil treatment were infected , however , symptoms of Verticillium wilt were found in only
3 of 5 plants at the 10• and 2 of 5 plants at the 15• treat~ents . Plants grown in soil held at both 25$ field capacity and flooded at 20• · and 25• C remained healthy .
Plants grown in soil held at air-dry , 50 and 75% moisture levels and 20• and 2s• C were infected as shown by isola­ tion and developed wilt symptoms . Only one of · five plants was infected in soil held at 30• C and 50$ moisture .
Table 11 shows that plants grown in soil held at 25% field capacity failed to produce symptoms even though infected .
In contrast all plants grown in the 50% moisture treatment showed wilt symptoms , and were , of course , infected .
Final temperature eabinet experiment
Results from the preliminary temperature cabinet ex­ periment were satisfactory except for variability within replications as well as from one sampling date to the next .
Variability was more pronounced in the lower than the higher temperature treatments . This variability was be­ lieved to be caused largely by an uneven suspension of microsclerotia and soil particles in the dilution medium and to a lesser extent by the sampling method . Steps were taken to reduce the variations by using CMC and standard­ izing the technique as described in the Materials and
Methods section .

TABLE 11. Isolation results from the temperature cabinet experiment showing effect of moisture and temperature on the population of Verticillium microsclerotia in soil as assayed by indicator plants. Soil samples were held for six months at indicated moisture and temperature before assa y ed.
Moisture level
Air-dry
25$ F. C.
50$ F. C.
75$ F. C.
Flooded
Symp Isola Symp Isola Symp Isola Symp Isola Symp Isola Symp Isola tom tion tom tion tom tion tom tion tom tion tom tion
+
+
+
+
+
+
+
+
+
+
+ +
+ +
+
+
+
+
+
+
+ +
+
+
+
+ +
+
+
+
+
+
+ +

39
With these modifications the experiment in the tem ­ perature cabinets was repeated with a change in one of the moisture treatments . Flooded soil was replaced by soil at field capacit y . The e x periment was started in Karch , 1 9 5 9 .
Samples were taken and analysis made from all treatments before they were placed in the respective temperatures .
Successive samplin g s were made at monthl y intervals for a period of 6 months .
Results as determined by soil dilution :
Data from this e x periment are presented g raphically in figures 5 to 1 0 and summarized in T able 12 . At soil temperatures of 5° , 1 0 ° and 15° C ( Figures 5 , 6 and 7)
Verticillium microsclerotia survived in all moisture levels for a period of 6 months . The rate of survival decreased slightl y as the te perature increased from 5° throu g h 15 ° c .
Microsclerotia survived fairly well at all moisture levels e x cept in soil at field capacity . Only in soil at field capacit y were numbers of microsclerotia reduced to less t han ten thousand per cubic centimeter .
The results from soils at 2 0 ° and 25° C were very similal' e x cept that the rate of reduction was more rapid at 25° . In soil held at 2 0 ° C (Fi g ure 8 ) Verticillium was not detected after a perio d of 5 months , with the e x ception of air-dr y and 25% moisture level . From the 25 ° C soil treatment ( Fi g ure 9 ) no Verticillium colonies were

40 present on dilution plates after a period of 5 months.
The rate of reduction was more pronounced in the 30°
C soil treatment than at lower temperatures (Figure 10).
Soil at field capacity held at 30° for 2 months contained no detectible Verticillium. At all other moisture levels there was a sharp reduction in the number of microsclerotia detected after 2 months, and viable microsclerotia were not detected after ~ months.
The trends observed in the preliminary experiment correspond closely to the results of the second experiment.
In both, best survival was obtained in soils at 50 and 75% moisture levels and low temperatures.
EFFECT OF HIGH TEMPERATURE ON SURVIVAL
Rapid reduction of microsclerotia within a short period was striking at 30° and 40° C. This effect was present in all moisture levels. To determine whether this reduction was gradual over a period of time, or abrupt as shown by long term sampling, an experiment was started in the temper­ ature tanks in September, 1959. Three moisture treatments consisted of air-dry, 50% field capacity and flooded. Each treatment was repeated three times at 30° and 40 ° C. Sam­ ples were taken just before the treatments were placed in the respective temperatures, and successive samples were taken three times a week (Monday, Wednesday and Friday) for a period of 35 days.

6
CAPACITY
(f) z
0
_j
-
_j
~
5
4
z
3
-
~
0 cr w
_j u
(f)
0 cr u
~
2
I
5° c
0 2 3
MONTHS
4 5 6
Figure 5. Effect of five levels of aoiature oD aurvival of Verticilliua aicroaclerotia iD soil held at 5• C for alx aoatha.
41

6
(/) z
0
_j
-
_j
~
5 z
4
I0°C
<I:
3
~
0 a:: w
_j u
(/)
0 a:: u
2
~
0 2 3
MONTHS
4 5 6
Figure 6. Effect of five levels of moisture on survival of Verticilliu• aicrosclerotia iD soil held at
10• c for six aonths.
42

6
(/) z
0
5
_J
_J
~ 4 z
<(
0 a:::
(.) -
2
0 a::: w
_J
(.)
(f)
3
0
FIELD CAPACITY
2 3
4
15° c
5 6
Figure 7. Effect of five levels of aoiature OD survival of V•~ticilliu• aicrosclerotia iD soil held at
15• C for six aoaths.
43

6
44

<1:
3
0
0:: u
-
2:
~
0
0:: w
_j u
(f)
2
6
(f) z
0
_j
_j
~
5
4 z
FIELD CAPACITY
25° c
2 3
4 5 6
Ftcure 9. Bffect of five level• of aoiature oa survival of Vertictlliua aicroaclerotia ia aoil held at
25• c tor aix aoatha.
45

6 en z 5
0
_j
_j
~ 4 z
3
<l:
1­
0
0:: w 2
_j u en
0
-
0:: u
~ fiELD CAPACITY
25%f·C·
75 •.4 f · C·
50%F·C ·
30°C
3
MONTHS
4
Figure 10. Effect of five levels of •olature oa survival of Yerticilliu• •ieroaclerotia iD soil held at
30• c tor alx •oaths.
46

TABLE 12. Percentage reduction of Verticilliua microsclerotia after a six month period in soil held at various levels ol moisture and tewperature.
Moisture levels 5• c
10° c
15° c
20° c
25° c
30° c
Air-dr y
2 5 % F. c.
50 % F. c.
75 % F. c.
Field capacity
75 . 0
5 7.
5
7 2.
9
7 3 . 6
99 . 8
8 1.2
8 2.5
5 4 .5
8 1.
4
99 . 9
83 . 3
8 7.1
88 . 8
95 . 8
99 . 9
1 00
99 . 8
99 . 9
1 0 0
1 00
1 0 0
99 . 9
99 . 9
1 0 0
1 00
1 00
1 0 0
99 . 9
100
1 0 0

48
Results as deter•ined by soil dilution:
The data are presented in Table 13 and figures 11 and
12. In the air-dry soil treatment held at 30° C (Figure
1 1) the initial level of 90,000 microsclerotia was reduced to 4 ,0 0 0 within 35 days. In the 5 0 % moisture level, 8.7 millions per cubic centimeter were reduced to 70,000; while in the flooded soil 8 .2 million were reduced to a level below detection within 12 days. In the lower two moisture levels reduction was gradual over 35 days, while in the flooded soil a sharp reduction was observed in less than 3 days.
At 4 0° C (Figure 12) reduction was much more rapid than at 3 0 °. Survival in the air-dry soil showed a grad­ ual reduction of microsclerotia over a period of 35 days.
In contrast a rapid reduction was evident in soil held at
5 0 % field capacity. Verticillium was not detected after
4 days. Similar •arked reduction was observed in flooded soil within 3 days with no detectable colonies at the end of 5 days.

49
TABLE 13. Effect of different levels of aoisture at and sclerotia. ao•
4o•
C on survival of Verticillium aicro­
of viable llicrosc lerot ia ( llillions Eer cc) so• c 4o• c
Days held Air-dry 50' F.C. Flooded Air-dry 50' F .C. Flooded
0 days 0 . 9 8 . 7 8 .2 0 . 9 5 . 7 8 . 4
3 d a ys 0 . 3
5 days 0 . 2
7 days 0 . 2
6 . 3
4 . 9
5 . 3
0 .1
0 .1
0 .1
0 . 2
0 .1
0 .1
0 .
2
0 . 0
0 . 02 o .
o o.o
0 . 0
10 days 0 . 01
12 days 0 . 02
1 days 0 . 01
17 days 0 . 01
19 days 0 . 02 2 . 5
21 days 0 . 01 2 .1 o .
o
0 . 001 o .
o o .
o
0 . 0 o .
o
0 . 01
0 . 01
0 . 01 o .
o o .
o o.o o .
o o .
o o .
o
0 . 002 o .
o o .
o
0 . 004 0 . 0 o.o
0 . 004 0 . 0 0 . 0
24 days 0 . 01 1.7
26 days 0.01 1.6
28 days 0 . 01 1.2 o .
o o .
o o .
o
31 days 0 . 01
33 days 0 . 01
0 . 9
0 . 7
35 days 0 . 004 0 . 7 o .
o o .
o o .
o
Percentage reduction 99 . 5 91 . 9 100
0 . 005 0 . 0 0 . 0
0 . 004 o .
o o .
o
0 . 003 0 . 0 o .
o
0 . 003 0 . 0
0 . 001 o .
o o .
o o .
o
0 . 001 o .
o o .
o
99 . 8 100 100

50
10
(/) z
0
_j
-
_j
~
8 z
6
4
1­
0
0:: w
_j
0
(/)
0
0::
-
0
~
4
2
50% FIELD CAPACITY
FLOODED
30° c
0 7 14
DAYS
21 28 35
Figure 11. Effect of three levels of aoisture on survival of Verticillium aicrosclerotia in Boil held at
30° C for 35 days.

51
10
(f) z
0
___I
-
___I
~
8 z 6
<(
1­
0
0::: w
___I u
(f)
0
0::: u
-
~
4
2
FLOODED
50% FIELD CAPACITY
40°C
0 7 14
DAYS
21 28 35
Figure 12. Effect of three levels of moisture on survival of Verticilliua aicrosclerotia in soil held at
40° C for 35 days.

52
DI SC USSION
T he data from these experi ents show the effect of soil moisture and temperatur e on survival of Verticillium microsclerotia . In general , microsclerotia survived poorly in soil at temperatures above 25 ° C . The effect of soil moisture is not so clear cut as the influence of soil tem ­ perature . There was , however , a sharp reduction in the microsclerotial population in flooded soil at all tempera ­ ture levels . Survival was best in soils held at mod e rate moisture levels and at low temperatures .
Reduction of the microsclerotial population as shown by indicator plants in soil held at S ° C was comparatively slight . At the end of six months the inoculum density was high enough to cause severe symptoms in peppermint plants grown in the experimental soil . The only exception was soil that was flooded , in which case the microscl e rotial population was reduced to a l e vel where symptoms produced in pepp e rmint plants were mild . Exc e pt in soils held at fi e ld capacity and flooded , differences in levels of soil moisture had only oderate effect on the survival of Verti­ cillium microsclerotia at 5 ° C .
Soil temperature of 10 ° C had an effect on the survi ­ val rate of microsclerotia similar to that observ e d at S 0
C . Here also the rate of reduction was gradual at all moisture l e vels . After six months the microsclerotial

53 population was still large enough to infect and produce symptoms in indicator plants , with the exception of soils held at field capacity and flooded moisture levels .
Comparable trends in the rate of Verticillium reduc ­ tion were obtained from soil that was kept at 15° and 20°
C. The rate of reduction in flooded soil , however , was more rapid at 20° than at 15° C (Table 6 and 7) . Also at
20° C the number of microsclerotia in soil held at 25$ field capacity was reduced below the level of infectivity
(Table 11) .
As soil temperatures were increased from 25° to 40° C a pronounced influence of temperature was evident as a great reduction in the number of surviving microsclerotia , irrespective of soil moisture levels . As temperature was increased the influence of soil moisture on the rate of survival of microsclerotia also appeared to be more pro­ nounced . In soil held at 25° C detectable survival was obtained only at moisture levels of 25 and 50% field ca­ pacity (Table 12) . The microsclerotial population was reduced to a level below detection after six months in soils held at other moisture levels.
Microsclerotia in soils held at 30° C decreased mark ­ edly within a period of two to three months . However , microsclerotia survived at a low level for six months in soil held at 50% field capacity .

54
Soil temperature of 4 0° C had great influence on the survival of Verticillium microsclerotia . At this high tem­ perature survival of microsclerotia in soil was limited to a short period . Only in air-dry soil did microsclerotia survive more than 5 days, and then only at a low level .
It is thus evident from the studies conducted to de­ termine the influence of high temperatures that Verticil­ lium aicrosclerotia can survive soil temperatures of 30° and 4 0° C for varying lengths of tiae, dependent on the moisture content of the soil. As one would expect 30° C was more favorable for microsclerotial survival . At the end of 35 days the microsclerotial population in the 30° C soil held at 50$ field capacity was large enough to cause severe disease in indicator plants. Contrary to this , the number of aicrosclerotia was reduced to a negligible level when soil was held 50% field capacity and 40• C for 3 days .
However, survival in air-dry soil was similar at both 30° and 4 0° C . At 30° C, soil moisture of 50$ field capacity seemed to have a moderating influence on temperature ef­ fect which was not seen at 40° c.
This is evident in
Figures 11 and 12 where microsclerotia rapidly disappeared in air-dry and flooded soil at 30° and 4 0° C, but at 30° C and 50$ field capacity survival was extended for more than
35 days.
These results strongly suggest that the effect of

55 temperature is more important in soils than the influence of moisture . This is in accord with the findings of Lud­ brook (20, p . 151) that temperature is the most important of the environmental factors influencin g the disease . It should be pointed out that soil temperatures of 30° to 4 0 °
C that have been found to be unfavorable to the survival of microsclerotia in this stud y correspond to the ran g e of temperatures that minimize infection in plants as demon ­ strated by different investi g ators (17 , 25 , 3 4 ) .
Schneider ( 34 , p . 1 4 2) found that Verticillium is
"active" in soil at all moisture levels above the wiltin g point . Results of the experiments presented in this thesis also su g gest that Verticillium microsclerotia would survive for lon g periods of ti e in all soil moisture levels e x cept flooded at temperature occurrin g naturally in the field .
Uicrosclerotial reduction in flooded soils may be attributed to the possible anaerobic conditions that are brou g ht about by sta g eant water .
The nature of the influence of temperature and mois­ ture on the survival of Verticillium microsclerotia in soil is not well understood . However , an explanation is attempt­ ed here .
The effect of soil temperature ma y be two-fold : 1) where the temperature has a direct lethal effect on the mi c rosclerotia in the soil which would be found only in

56 the higher temperatures ran g ing from 30° C upwards . 2) where th temperature bas an effect on the germination of microsclerotia in the soil , or upon the mycelium produced by the germinated microsclerotia . Temperature requirements for the germination of microsclerotia in the soil are not found in the literature . It can be assumed , however , that temperatures below 15° C will not be favorable for micro­ sclerotia! germination . The optimum range would probably fall between 20° and 30° c .
Nelson (25 , p . 151 , 155) in his experiments with peppermint plants found that the ran g e of temperature critical for infection of plants in the field was between 22° and 28° c .
He also observed that soil te peratures below 20° C were unfavorable for infection in the field . Since the data presented here show that the population of microsclerotia falls rapidly when temperature is increased from 25° to 4 0° c , it is only natural to believe that survival rate of germinate d microsclerotia would also fall rapidl y . On the other band , the germinated microsclerotia in the lower temperatures may colonize the or g anic matter in the soil for a short period and produce more microsclerotia when available food is exhausted. If this explanation is found to be true the influence of soil moisture on the survival of microsclerotial populations may also be two-fold : 1) a direct effect on the g ermination of microsclerotia in the

57 soil . 2) moisture acting as a factor limiting the influ­ ence of temperature . Moisture is required for the germi ­ nation of most fungus reporductive units in the soil . The moderating influence of soil moisture is evident in soil held at 30 ° C with 50% moisture content ( Figure 11) . The population of microsclerotia in air dry and flooded soil at 30 ° and 40 ° C showed equal survival rates . In soil held at 30 ° C and 50% moisture a large microsclerotial population survived more than 35 days while at 4 0° C and
50% moisture microsclerotial survival was limited to a period of 3 days . Thus , at 4 0 ° C this moderating effect was eliminated by the lethal effect of temperature .
A remarkable effect of moisture in increasing the apparent microsclerotial population was noticed in almost all treatments throughout this study . It is evident from the data in Tables 4 , 5 , and 6 and Figures 5 and 6 that the counts of Verticillium colonies from the second samp­ lings were higher at 25% and higher moisture levels than the original number of microsclerotia added to the soil .
This increase was general in all moisture treatments and was especially pronounced in soils with 50% moisture and above . Even though there was no availabl e explanation for this phenomenon , subsequent experim e nts showed it to be of a purely physical nature . A batch of inoculated air dry soil was divided into two parts nd water was

58 added to one part to bring the moisture level to 50$ field capacity. Samples were taken from both parts immediately.
Successive samples were taken at 2, 5 , 12, 2 4 and 4 8 hours.
All samples were immediately assayed by soil dilution to determine the number of microsclerotia. The two treatments were held at 5° C during the experiment. From the samples that were taken immediately after the addition of moisture twice as any Verticillium colonies wer e obtained in wet soil as in the air-dry treatment. There was a gradual in­ crease in counts from wet soil with an increase in time from zero hour to 2 4 hours. The number of Verticillium colonies obtained from air-dry soil remained almost con­ stant over this period. Similar results were obtained in later experiments. The data from these experiments suggest that pre-wetting the soil may increase the number of Verti­ cillium colonies on soil dilution plates : 1) by separating pre-moistened microsclerotial clumps in the process of making dilutions, 2) by breaking clumps of soil and dis­ persing "trapped" microsclerotia into the dilution medium, and 3) by preventing the formation of air-pockets which trap microsclerotia and soil particles in one clump.
Earlier investigators proved that Verticillium would remain in a field for a long period of time in the absence of plants (19, 25, · 5). Rotations with non-susceptible crops did not control Verticillium wilt in different hosts

59
(19 , 25) . This long persistence in soil has been attri­ buted to the resistance of microsclerotia to the environ­ mental factors . Hence , in the absence of a host , the fun ­ gus would be dependent on microsclerotia to survive .
Keyworth (13), Wilhelm ( 44 ) and Tolmsoff (39, p . 112) clearly de onstrated that the severity of infection is related to inoculum density of the fungus in soil . It is evident from the data presented in this thesis that soil moisture and temperature are two important factors in the environment that influence survival of Verticillium micro­ sclerotia .
The application of this information for the control of Verticillium wilt may necessitate further studies in the field . It may not be possible , however , to eliminate the fun g us fro a soil , but proper manipulations of soil moisture and temperature would tend to reduce the inoculum densit y to a level where disease incidence would be minimized .

60
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64