Some factors affecting survival of sclerotia of Macrophomina phaseolina in soil

Sorl Bid. Btochem. Vol. 9, pp. 337 to 341. Pergamon Press 1977. Pm&d

in Great Brimin.

SOME FACTORS AFFECTING SURVIVAL OF ~ACROPHO~~~A PHA~~OLI~A G. C.

OF SCLEROTIA IN SOIL

PAPAVIZAS

Soilborne Diseases Laboratory, Plant Protection Institute. Agricultural Research Service, US. Department of Agriculture, Beltsville, MD 20705, U.S.A.

least 757; of the sclerotia of Macrophomina phaseolinu survived for 1 yr in most natural soils kept at 26°C and at 5Cr55% of the soil moisture holding capacity (m.h.c.). Although survivability was reduced in a very acid soil (pH 4.5) collected under a pine stand, 33% of the sclerotia survived for 1yr. Soil pH had very little or no effect on sderotial survivability. Of three organic amendments tested (alfalfa hay, chitin, pine needles) only ground alfalfa hay at 0.8% (w/w) reduced s~lrvivabil~ty of sclerotia in soil by about 75% in a year. Alfalfa hay at 0.4:/, reduced survivabiIity by 36%. Various N sources added at 200 pgNg- ’ soil had no effect on survival. Of 13 fungicides tested, only benomyl and captan at 20 pg a.i. gg’ soil appreciably reduced populations of sclerotia in soil. Soil temperature and moisture content were the two most important factors affecting survivability of sclerotia. At -5 or 5°C the biggest drop in sclerotial survivability occurred when the soil was incubated moist (at 50% m.b.c. or more). At 26°C the biggest drop occurred in air-dried soil (2-3% m.h.c.) and survivability was decreased to some extent at 15 and 30% m.h.c. Survivability also dropped rapidly in moist soil (SO--55%m.h.c.) exposed to four cycles each having 3-week freezing (- 5°C) and 1 week thawing (26°C). Sclerotia in air-dried soil (2-3% m.h.c.) continuously kept at -5°C maintained nearly complete survivability after 16 weeks. Sclerotia survived almost SO-900/, in moist soil (50-55x m.h.c.) kept for 16 weeks at 26°C or in moist soil exposed to four cycles each having 3-week thawing (26°C) and l-week freezing (-5°C). Summary-At

Macropkomina phaseolitw (Tassi) Goid. [Rkizoctonia hataticola (Taub.) Butler] causes root rot, charcoal

rot and seedling disease on more than 400 plant species (Reichert and Helling, 1947). There is now abundant evidence that the pathogen survives in soil as small, black sclerotia (60-100 x 56-80 pm) formed on colonized host tissue and liberated into the soil matrix upon tissue decomposition (Cook et al., 1973; Ilyas and Sinclair, 1974; Meyer et al., 1973; Papavizas and Klag, 1975; Smith 1969a). The importance of sclerotia in the epidemiology of this root pathogen has also been stressed (Ilyas and Sinclair, 1974; Norton, 1953; Smith, 1969a,b; Watanabe et (II., 1970). Very little work has been done on factors affecting survival of M. phuseolina sclerotia in the laboratory and even less in the field. Cook et al. (1973) found that some sclerotia survived in sorghum and corn stalk tissue for 16 and 18 months, respectively. During that period, germination of sclerotia decreased from 80 to 23”/, in one field and 59 to 31% in another. In the same study, germination of sclerotia obtained from soil was only l--S%. Ghaffar and Akhtar (1968) observed that sclerotia of M. pkaseolina survived for at least 10 months in infected cucurbit roots under dry storage in the laboratory. Survival was reduced to only 15 days in root segments buried in moist soil. Sclerotia on fiberglass survived more than 3 months at various soil moisture contents (Ghaffar, 1968). In a recent study, sclerotial survival in colonized basal stem segments of the soybean cultivar Amsoy 71 decreased as soil moisture and length of exposure increased (Dhingra and Sinclair, 1974). The greatest survival was in segments buried in air-dried soil and the

least in those buried in soil kept at 80 or iOO%m.h.c. Also in these studies, survival decreased more in segments buried in soil amended with glucose and NaNO, at high C:N ratios than with the same amendments at low C:N ratios (10 or 20). All the experiments in this study were performed at 30°C and the final samples were removed from soil after 7 weeks. Almost all recent studies on survival were performed with tissue- or medium-formed sclerotia in the laboratory. Watanabe (1973) showed that viability of tissue- and soil-formed sclerotia gradually declined in soil, but some sclerotia were still viable after 4 yr of soil exposure. Recent studies in Illinois (Dhingra and Sinclair, 1975) showed that ger~nability of M. phaseolbta sclerotia dropped rapidly in soil maintained at 30°C and at high moisture (60, 80 or 100% m.h.c.). No reduction was observed at 0% m.h.c. and 4&500/, reduction occurred at 20 and 4Ooj, m.h.c. These studies, however, were performed for 7 weeks only. Bristow and Wyllie (1975) indicated that freezing and thawing moist soils artificially or naturally infested with sclerotia resulted in 90 and 50% reduction in sclerotial germinability, respectively. Freezing and thawing eight times of air-dried, artificially infested soil resulted in less than 15% reduction. The present studies were undertaken to evaluate for prolonged periods the importance of soil factors (temperature, moisture, pH, organic and inorganic amendments, fungicides) on survival of free sclerotia in soil.

337 S.&R. 9;5-c

MATERIALS

AND METHODS

M. pkaseolina isolates Mp-1, Mp-3 and Mp-19 were used throughout. Sclerotia were produced in the laboratory as described previously (Papavizas and

Klag. 1975). After the sclerotia were dried at room temperature, they were passed through a 177-itm sieve (80-mesh) and individual sclerotia that passed the sieve were used in all studies. The soils used were Elsinboro sandy loam (ESL). pH 5.0; Gaiestown-Evesboro sandy loam (GESL). pH 4.8; Sassafras loamy sand (SLS), pH 6.4: Rumsford sandy loam (RSL), pH 4.5; Illinois clay soil No. I, pH 6.2; and Illinois clay soil No. 2. pH 7.2. In some instances. the pH of the soils was adjusted to higher values. The pH was determined el~trometrically in mixtures of soil and water fl part soil:2 parts water, w/w). Air-dried soils were passed through a S-mm sieve. mixed thoroughly, and stored dry. The soil water content was determined by the gravimctric method with oven drying and was expressed as percentage of water in the sample on a dry-weight basis (Gardner. 1965). In the ~~rnendnlent experiment, chitin and dry, ground alfalfa hay and fallen pint needles were added to ESL infested with sclerotia of isolate Mp-3. The amendments were added at 0.4 and OX‘?; (w/w) and the amended soils were kept moist (45-5O”i MHC) at 26-28!8’Cfor 52 weeks. To test the eI%cts of various systemic and nonsystemic fungicides on sclerotial survival in soil, t,4-dichloro-2,5-dim~thoxybenzene (chloroneb), 5,6-dihydro-Z-methyl- 1,4-oxathiin-3-carboxanilide (carboxin): methyl l-(butylcarbamoyl)-2-benzimidazolecarhamate (henomyl): dimethyl[( I.?.- phenyIene)bis(iminocarbonothioyl)j bis[ carbamatef (thiophanate methyl); tetr~~chlorois~~phthalonitrile (chloroth~~lonil)~ hi- [(trichloromet~lyl)thio] - 4 - cyclohexcne - I,2 - dicarboximide (captan); bis(dimcthylthio~~rbai~oyl)djsulfide (tl~iram); ~-(4”thiazoiyl)benzimidazol~ (thiab~ndazole): pentachloro~litr~~henzene (PCNB); PCNB + 5~thoxy-3-(trichioro~~et~~yl)-I,2,4-thiadi~~ole (FCNB + ETMT); fungicide MF-586 (IS”, thiophanate methyl + 60% maneb); Banrot( 1SJ’;, ETMT + 25y;, thiophanate methyl); and experimental fungicide RH-3928 were added to sclerotia-infested soil at 20 ,~g a.i. g- ’ soil and the treated soils were incubated at 5OY;,m.h.c. and 26-28°C for 52 weeks. In all experiments, amended and control soils were subdivided into 200-g portions and these were placed in 4OO-mlbeakers. The beakers were weighed, covered

KESl LTS

Six soils infested with sclerotia of isolates Mp-3 and Mp-19 (used separately) were brought to X1”,,m.h.c. and incubated at 26 ‘C for 1 yr. Assays for surviving sclerotial populations were performed after 7. IX. 30 and 52 weeks. With isolate Mp-f9, 75’?,,or more of scierotia survived in five of the six soils tested (Fig. 1). Thus, of a total population of 200 s&rot& g _ ’ soil at zero time, 1% sclerotia g ‘-I soil or more were recovered after 52 weeks. Although populations declined to fess than IOOg-’ soil in RSL (pH 4.5). a soit collected under a Virginia pine stand, the survival rate was 33”‘~;, in this soil. With minor exceptions, sclerotia of isolate Mp-3 survived in a similar manner in these moist soils. In another experiment, two acid soils were used. Samples of ESL fpH 5.0) were adjusted with Ca(OHf, to pH 6.X and 7.5; and GESL (pH 4.8) to 6.3 and 7.2. Forty per cent of sclerotia of isolate Mp-3 survived in ESL. at pH 5.0 after I yr and about 6Ol!d survived in the same soil at pH 6.8 and 7.5. About h@‘,% of Mp-3 sclerotia survived in GESL after I yr. regardless of soil pH. Isolate Mp-19 behaved similarly. cfl&t 0f’oryurfic mew/fwf7ts urtd ffitroyrfz off swcivul Of the three organic amendments tested. only alfalfa hay at OX’!,,reduced scferotial survival in soil by about 751;, after 52 weeks (Table 1). Alfalt:d hay at a rate of U.&&,reduced sclerotia by 36”,, in 1 yr. Chitin and pine needles were in&ective. Pine needles were used because of the reduction of s~irvivability observed in an &id soil collected from a pint stand. To determine the effect of N on sclerotial survival in soil, NdNt&, &NO,. 4H,Q, WI&SO,, (NH~)~HPO~ an~~~onjLln1 polyphosphate (15:62:0). ammonium polyp~~~sphate (liquid I I :37:(f). and urea suspension (32:O:O) were added to ESL, previously adjusted to pH 7.2. at a rate to provide 200 pg N g- ’ so& The soil had been previously infested with scterotiu of isolates Mp-1 and Mp-3, used separately. Assays for scteroriat l?~~pulations performed after 9.

v&h ~ous~llo~d pbWic Wrap permeabk to gases othel than water vapor. and incubated at various temyera-

tures specified in each particular experiment. The beakers were weighed once per month and, when necessary, additional water was added to bring the moisture content to the desired amount. Immediately after water addition, the soils were mixed th~ro~lghly and the beakers were covered. With this procedure, less than 4% of water was lost each month. At zero time anti at intervals up to 51 weeks, IOg soil (dry weight basis) was removed from the beakers and assayed for surviving sclerotia. Populations of sclerotia were assayed with a special dilution-plate method on potato dextrose agar ~Dexon-oxgall-rose bengal (PDA-DORB), a selective medium (Papavizas and Klag, 1975). The dilution plates were incubated at 30°C and colonies of n/l. phusrolin~l were counted after 6 days. The number of colonies counted from each gram of soil correspond to the numbers of surviving single sclerotia in soil. Four replications were used throughout.

. .. . .., lirrcrin six moist soils incubated at 25’c for 51. weeks. A. Illinois clav soil No. I : B, Illinois clay soil No. 2’ f’ S&aafras loamy sand; D. Galestown-Eveshro sandy Ida; (pH4.X); E, Elsinboro sandy loam (pH 5.0): F. Rumsford satdy loam JpH 4.5).

Survival of M. phaseolina

339

Table 1. Effect of organic amendments on survival of sclerotia of Macrophomina phaseolina (isolate Mp-3) in soil as determined by the dilution plate method No. of surviving sclerotia g-r soil* 36 weeks 52 weeks 9 weeks

Amendment None (control) Chitin 0.4% 0.8% Pine needles 0.4% 0.8% Alfalfa hay 0.4% 0.8%

630 At

530 A

450 A

620 A 530 B

480 A 440 AB

460 A 420 A

630 A 550 AB

470 A 450 AB

400 A 410 A

530 B 290 C

320 BC 190 c

290 B 100 c

* No. of sclerotia at zero time was 68Og-’ soil. t Treatments having similar lettering per assay time do not differ at P = 0.05. 20,36 and 52 weeks showed no differences in survival of the two isolates in the soil portions amended with the various N sources. About 7580% of the sclerotia survived all treatments. Effect of fungicides

on survival

Of 13 fungicides tested with isolate Mp-1, only benomyl and captan appreciably reduced survival of sclerotia in soil (Table 2). Only nine fungicides were included in Table 2. Captan and benomyl reduced sclerotial populations in soil by 83 and 60x, respectively, in 15 weeks of incubation. The effectiveness of benomyl appeared to increase somewhat when added to soil together with thiram (70:30), but the difference was not statistically significant. The other fungicides were ineffective. EfSect of temperature and moisture on survival Air-dried soil (2-3x m.h.c.) and moist soil (SOo/,m.h.c.) were infested separately with sclerotia of ISOLATE MQ-IS

SOIL AT Z-S%MHC

200 -

j

k-r:

02

I6

24

36

52

WEEKS

Fig. 2. Populations of sclerotia of Macrophomina phaseolina in soil (isolate Mp-19) as affected by soil moisture, temperature, and length of incubation.

I

I

1

1

6

IO

36

s2

WEEKS

Fig. 3. Populations of sclerotia Macrophomina phaseolina (isolate Mp-13) in soil as affected by various soil moistures.

Mp-3 and Mp-19 (240 sclerotia g-’ soil). The airdried and moist soil samples were subdivided into five portions and four replications of each were placed at -5, 5, 15, 25 and 35°C for 1 yr. Ten-gram samples from each replication were removed after 2, 16, 24, 36 and 52 weeks and assayed for surviving sclerotia. Survival of sclerotia differed considerably with variation in temperature of incubation of soils, but this effect depended on the soil moisture content (Fig. 2). With air-dried soil, survivability of sclerotia of isolate Mp-19 declined rapidly at 15,25 and 35°C. No sclerotia survived after 36 weeks at 35°C and after 52 weeks at 25°C; very few survived at 15°C. About 40-45x of sclerotia survived after 1 yr in the air dry soil at -5 and 5°C. With moist soil kept constantly at 50% m.h.c., the effect of temperature on survival was reversed. About 6&65x of sclerotia survived in the moist soil kept at 15, 25 and 35°C for 1 yr; 2&24x at 5°C; and only about 2% at -5°C. Almost identical results were obtained with isolate Mp-3. There was considerable interaction between soil moisture and temperature on sclerotial survival. Therefore, an experiment was performed to study the effect of various soil moisture regimes on survival. Portions of air-dried ESL (pH 7.0) containing sclerotia of isolate Mp-19 were moistened to 15, 30, 50, 70 and 90% m.h.c. Four replications of each moisture treatment were incubated at 5°C and another four at 26°C. At 5°C the greatest survival was observed in airdried soil and in soil’kept at 15% m.h.c. (Fig. 3). Survival dropped considerably when the soil was kept at 30, 50, 70 and 90% m.h.c. With the high moisture contents, drop in survivability became pronounced after the 18th week of incubation, and with 30% m.h.c. after the 36th week. At 26°C the biggest drop in sclerotial viability occurred in the air-dry soil and at 15 and 30% m.h.c; the smallest decline occurred at 50, 70 and 90% m.h.c.

Table 2. Effect of fungicides on survival of sclcrotia of .MuoophorttirlLI phseolit~tr (isolate Mp-I ) in soil as determined by the dilution plate method No. of surviving sclerotia g- ’ soil+ 6 weeks 15 weeks

Fungicide* None (control) Chloroneb Chlorothalonil Carboxin RH-3Y28 Thiram Thiophanate methyl

550 520 450 5tt) 450 430 360

Benomyl

190 CD

170 BC

I20 D 180 CD

100 c 70 c

Benomyl Captan

+ thiram

A; A A A A AB ABC

420 A 410 A 300 A 360 .A 3YO A 370 A 370 A

to soil at 20 mg a.i. kg- ’ soil. at zero time was 600 g ’ soil. : Treatments having similar lettering per assay time do not differ at P = 0.05. * Fungicides

added

+ No. of sclerotia

Results obtained at 907; of the soil m.h.c. were similar to those at 7O”a. Because sclerotia did not survive in moist soil kept at -5’C for more than 16 weeks (Fig. 2), an experiment was performed to find out whether prolonged freezing is needed for killing the sclerotia or whether the same effect can be obtained by shorter alternate periods of freezing and thawing of the soil. ESL was adjusted to pH 7.8. kept moist for 2 months and airdried. The series of treatments consisted of: Air-dried soil (2-3% m.h.c.) and moist soil (50-55X, m.h.c.) kept continuously at -5’C for 16 weeks; and air-dried and moist soil kept for 3 weeks at -SC and then transferred to 26°C for 1 week. At the end of the l-month cycles, assays were performed for surviving populations of sclerotia and the soil portions were transferred to -5°C to begin the cycle again. The second series of treatments included: Air-dried soil and moist soil kept continuously at 26°C for 16 weeks; and the same kind of soils kept for 3 weeks at 26°C and then transferred to -5°C for I week. At the end of each l-month reversed cycle. dilution plate assays were performed. Sclerotial survivability dropped rapidly in moist soil kept continuously at -5’C or exposed to a cycle of 3 weeks freezing and 1 week thawing (Fig. 4). Sclerotia in air-dried soil at - 5’ maintained their viability almost intact. Sclerotia in air-dried soil exposed to the freezing-thawing cycle began losing their viability after 8 weeks (two cycles of incu~tion). In the reversed cycle the results were the opposite. Sclerotia in air-dried soil lost their viability rapidly after 8 weeks, irrespective of whether they were kept continuously at 26°C or in a cycle of 3 weeks at 26°C and 1 week at -5°C. Sclerotia in moist soil kept continuously for 16 weeks at 26C or exposed to a cycle of 3 weeks at 26°C followed by 1 week at -5°C survived almost 80-90”; up to 16 weeks. DISCUSSION

Of various factors tested, only alfalfa hay at a relatively high rate (0.8”/;, w/w) reduced survivability of sclerotia of M. ~~~~z.s~o~jtzf~considerably after 52 weeks

of incubation. The inability of chitin. pine needles. various soil pH reactions and several N sources to reduce appreciably sclerotial numbers in soil indicates how resistant these propagLlles are and stresses the well known fact that sclerotial fungi are very difficult to control or reduce by inorganic and organic amendments. The ineffectiveness of pine needles was disappointing, especially since there was some reduction in viability when sclerotia were incubated in soil from a pine stand. The ability of alfalfa hay and, to a lesser extent, barley straw anlend~nents to suppress cotton root rot caused by M. p~us~o~~~~~~ was observed by Ghat%r et al. (1969). In their studies, suppression of disease but not necessarily reduction in sclerotial inoculum density. was attributed to an increase in bacteria and actinomycctes antagonistic to M. p/trrsc~oli~. The ineffectiveness of the N fertilizers to increase or decrease sclerotial viability, and consequently sclerotial populations in soil, was completely unexpected. Dhingra and Sinclair (1975) found that NaNO, alone reduced sclerotium populations the first 2-3 weeks; then by the 7th week, NaNO, increased populations above those in control soil. The differences observed between the results with N of the present study and those of Dhingra and Sinclair may be due to the fact that they used 0.94 or soil, whereas only 0.2 mg N g-’ soil i.@mgNg-’ were used in the present study.

-

I andlI: Can&sly at -5’C mand1E:3wbsat-5’E,iwkat26°C (FourGYCW

DRYSOIL

.--------- lOISi SDlL

WEEKS

._ ---*-----b -

---------.

i a co

Ita

T D i c

300

ORVSOIL MOIST SOIL

-._-*.._______

-

I nd 1: Continuaurly at ZE’C ~and~3Wks4126~C.Iwkat-SnC

-__--------______________.

WEEKS

Fig. 4. Populations of sclerotia of Mucrophotnitxt phaseolim in soil as affected by soil moisture and monthly cycling of temperature. Upper figure, cycle beginning at -5-C: Lower figure, cycfe beginning at 26°C.

G. C. PAPAV~ZAS

Although considerable work has been done on the efficacy of various fungicides to control diseases caused by M. phaseolina (Al-Beldawi et al., 1973; Moustafa and Wyllie, 1975; Shanmugan and Govindaswamy, 1973; Singh et al., 1973; Vir, 1974), very little research has been done on direct fungicidal effects on sclerotia in soil. Recently, Ilyas et al. (1975) found that benomyl was the most effective of fungicides tested in reducing sclerotial populations in soil. In the present experiments, benomyl and captan reduced sclerotial numbers in soil. The magnitude of reduction, however, at the fungicidal rates used (20 mg a.i. g- 1 soil), was not impressive. Even with captan, the most effective material, 17% of sclerotia survived the treatment after 15 weeks of incubation. This indicates that eradication of sclerotia of this pathogen from soil may be very difficult, or even impossible, with acceptable, reasonable rates of fungicide applications. Soil moisture and temperature were the two most important factors aflecting viability of sclerotia of M. phaseolina in soil. The following four moisture-temperature combinations appeared to be detrimental to sclerotial survival in soil: (i) Low soil moisture (airdried soil at about 2-3”! m.h.c.) and high temperatures (25 and 35°C); (ii) high soil moisture (S&90% m.h.c.) and low temperatures (-5 and 5°C); (iii) high soil moisture (50xm.h.c.) and a cycle of freezing for 3 weeks and thawing for 1 week and (iv) low soil moisture (air-dried soil) and a cycle of 3 weeks at 26°C and 1 week freezing (-5°C). The best survival of scierotia for 16 weeks was obtained with moist soil at 26°C and with air-dried soil at -5°C. The detrimental effect of the high soil moisture combined with freezing temperatures may be attributed to ice crystal formation in the sclerotia as postulated by Bristow and Wyllie (1975) in similar experiments. The destructive effect of air-dried conditions and high tem~rature may be explained by assuming that protracted desiccation at high temperature irreversibly damages the protoplasts of individual cells in the sclerotial mass (Papavizas, unpublished data). The detrimental effects of high soil moisture at low temperatures and that of low soil moisture at high temperatures may be applicable in the field for cultural control of M. ~~aseoli~l~~. AcknowEedgemmt-I thank N. G. Klag for assistance and for the preparation of the graphs.

341

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NORTON

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