Survival of sclerotia of Macrophomina phaseolina and Sclerotium cepivorum after drying and wetting treatments

Exposure of sclerotia of Macrophomina phaseolina to 0 and 33% relative humidity (r.h.) for 12 weeks and of Sclerotium cepivorum to 0, 33 and 55% r.h. for 20 weeks did not reduce their germinability on agar. Exposure to 78% r.h. caused high loss of germinability in M. phaseolina and complete loss in S. cepivorum. After 7-day exposures respective moisture contents of sclerotia of M. phaseolina and S. cepivorum were 1 and 2% at 0% r.h.; and 10 and 14% at 78% r.h. M. phaseolina sclerotia held at 0% and 33% r.h. in desiccators for several times up to 12 days did not decrease in subsequent survivability in moist soil, unlike sclerotia held at 78% r.h. for 4 days.More sclerotia of M. phaseolina were colonized by fungi and Streptomyces spp. on alkaline soil than on acid soil. On alkaline soil twice as many sclerotia were colonized after exposure to 0% r.h. as after exposure to 33, 55 and 78% r.h. Colonization of S. cepivorum sclerotia was as high on acid as on alkaline soil and 3 times as high on sclerotia treated at 0% r.h. as on those treated at higher r.h. Attempts to ascertain the effects of colonization on sclerotial viability were unsuccessful. Incubation of sclerotia of M. phaseolina in moist Rumsford sandy loam (50% m.h.c.) for 20 weeks reduced survivability by 43%. At room temperature, alternate drying and wetting of soil containing sclerotia did not appreciably affect survivability of either pathogen. Survivability of S. cepivorum sclerotia was highest when the sclerotia were incubated in air-dried soil (2–3% m.h.c.) for 20 weeks.Incidence of white rot on onion seedlings transplanted to S. cepivorum-infested soil was higher in soil that had been air-dried for 20 weeks than in soil that had been alternately wetted and dried. Sclerotia that were exposed to 0% r.h. for 7 days before soil incubation produced little white rot.     

Polyethylene mulching of soil to reduce viability of sclerotia of Sclerotium oryzae

Mulching of Sclerotium oryzae infested soil (moist or dry) with polyethylene sheets during hot summer days of May and June increased the soil temperature at 5 cm from 36°C (unmulched) to 48°C (wet) and from 44 to 52°C (dry) and at 20cm from 32 to 38°C (wet) and from 35 to 39°C (dry). In artificially-infested soil, the sclerotia were not eradicated but 95–100% loss in viability was observed at 5 cm by a mulch treatment for 1 week and at 20 cm by mulching for 8 weeks. Mulching effects were not influenced by moisture content of soil or by amendments with lucerne or wheat straw. Mulching of naturally-infested soil at a second site did not eradicate S. oryzae but reduced sclerotial viability by 93%.     

Carbon loss by sclerotia of Sclerotium rolfsii under the influence of soil pH,temperature and matric potential and its effect on sclerotial germination and virulence

Germinability and virulence of sclerotia of Sclerotium rolfsii were assessed after 50 days of exposure of ¹⁴C-labeled sclerotia to soil at 0, −5 and −15 kPa and pH 6.9, or to soil at 15, 25 or 30 °C, pH 5 or 8 and −1 kPa. Evolution of ¹⁴CO₂ accounted for the greatest share of endogenous carbon loss from sclerotia under all soil conditions, except in water-saturated soil (0 kPa), in which sclerotial exudates contributed the major share of carbon loss. Total evolution of ¹⁴CO₂ from sclerotia in soil at −15 kPa (42.4% of total ¹⁴C) and at −5 kPa (38%) was significantly higher than at 0 kPa (23.8%). Evolution of ¹⁴CO₂ in soil at 25 or 30 °C was more rapid than at 15 °C with regardless of pH. Loss of endogenous carbon by sclerotia was the greater after 50 days of exposure to soil at 0 kPa, or at 25 or 30 °C and pH 8, than at other soil conditions. Sclerotia exposed to water-saturated soil (0 kPa) showed a more rapid decline in nutrient independent germinability, viability and virulence, than to those exposed to −5 or −15 kPa. Sclerotia became dependent on nutrient for germination and lost viability and virulence within 30–40 days in soil at 25 or 30 °C, pH 8. However, more than 60% of sclerotia retained viability in soil at 15 °C regardless of pH, even after 50 days. Radish shoot growth was increased significantly by the sclerotia that had been exposed to soil at 0 kPa, or to soil at 25 or 30 °C and pH 8 for 50 days. In conclusion, carbon loss by sclerotia during incubation on soil at different pH levels, temperatures and water potentials was inversely correlated with sclerotial ability to infect radish seedlings. The relationship between carbon loss by sclerotia and radish shoot length was positive.     

The effect of dry conditions on subsequent leakage and rotting of fungal sclerotia

Dried sclerotia of Sclerotium delphinii rotted in moist soil whereas those of Sclerotium cepivorum. Botrytis cinerea and Botrytis tulipae did not. A number of fungi invaded dried sclerotia of S. delphinii in soil, the principal coloniser found in the first sampling being Trichodermu hamatum. Leakage of ¹⁴C compounds from dried labelled sclerotia placed in water was rapid and was little affected by variation in leakage temperature from 1 to 25°C or by prolonging the drying period beyond a day. Leakage from dried sclerotia which were allowed to imbibe water through a small part of their surface was much reduced. Sclerotia which were redried after leakage leaked again when returned to water but with all four fungi the first of three leakage cycles gave the highest ¹⁴C levels. Loss in dry weight in the first leakage cycle was greater with S. delphinii than with the other three fungi and this may be related to the poor survival of dried sclerotia of S. delphinii in moist soil. Substances lost during leakage appear to originate from within sclerotial hyphae rather than from the hyphal free space.     

Diallyl-disulphide to reduce the numbers of sclerotia of Sclerotium cepivorum in soil

A single injection of 0.2 ml diallyl disulphide (DADS) at 0.156% (v/v) into soil containing naturally-produced sclerotia of Sclerotium cepivorum and maintained in the laboratory at 15°C stimulated sclerotial germination and reduced sclerotial numbers by 67%; ungerminated sclerotia remained viable. Higher concentrations of DADS had no additional effect except that at 20% (v/v), germination was slightly inhibited. A similar reduction in sclerotial numbers was obtained when the mixture of soil and sclerotia was exposed to DADS vapour. Four, monthly applications of DADS at 0.2 ml 0.15% (v/v) per application did not give a further reduction.The effect of DADS was temperature dependant, with a reduction in sclerotial numbers of 65 and 9% at 15 and 5°C respectively.     

Time-temperature relationships for the inactivation of sclerotia oF Sclerotium oryzae

The viability of sclerotia of Sclerotium oryzae was tested in both wet and dry soil under simulated elevated temperatures in either constant, cyclic or short temperature regimes. In wet soil viability was reduced to zero by a constant temperature of 45°C and above for 1 day or a minimum oven temperature of 55°C for 2 h. Viability of sclerotia were destroyed by either a 10-day constant temperature of 40°C or a 2-h temperature cycle for 3 days at 50°C or 12 days at 45°C. Below 40°C sclerotia were not affected. In dry soil at 60°C, a 3-day constant temperature or a 12 days 2-h temperature cycle eliminated sclerotial viability. Data on time and temperature relationships on loss in viability of sclerotia in soil could thus be used to predict efficacy of soil solarization.     

Reduction in viability of sclerotia of Macrophomina phaseolina with polyethylene mulching of soil

Mulching of Macrophomina phaseolina-inksted soil (moist or dry) with transparent polyethylene sheets during the hot days of May increased temperature of wet soil at 5 cm from 37°C (unmulched) to 52°C (mulched) and of dry soil from 52°C (unmulched) to 65°C (mulched). At 20 cm mulching increased temperature from 30°C to 41°C (wet) and from 38°C to 42°C (dry). In artificially-infested soil. the sclerotia of M. phaseolina were eradicated at 5 cm by a mulch treatment for 1 week and at 20 cm depth 50% sclerotia lost viability in wet soil but were not affected in dry soil. In a naturally infested soil (5–7 sclerotia g^?1), which gave 20% infection on Vigna, the sclerotia were reduced to such an extent that after 1 week mulching no disease was observed on Vigna.     

The response of cylindrocladium conidia to soil fungistasis

Direct observation of washed conidia of Cylindrocladium scoparium on non-sterile soils, air dried and rewetted immediately before deposition of conidia, indicated that peak germination (33–58%) occurred after 24 h incubation at 26°C. Peak germination on continually moist soils was lower (18–26%) than on rewetted soils. Lysis of germ tubes and germinating conidia on continually moist soils at 26°C was evident with 48 h. Conidia did not germinate on continually moist soils at 6°C and lysis did not become apparent until 168 h. Conidia germinated at a high level (93–99%) in axenic culture in the absence of exogenous C and N sources. The inhibition of conidial germination on soils may be attributed, in part, to the presence of soil volatiles. Germination of conidia placed on washed agar disks and exposed to volatiles from four soils ranged from 51 to 86% of the no-soil controls. Addition of carbon (13 ng C per conidium as glucose) and nitrogen (65 pg N ng^?1 C as NH₄C1) nullified the inhibitory effect of the soil volatiles. Germinability assayed on a selective medium at 26°C of conidia in artificially infested soils (approximately 10⁴ conidia g^?1 soil) decreased progressively during incubation at 26°C from 1 week to 4 months. No germinable conidia were recovered from artificially infested soils after 2 months incubation at 6°C. Conidia of C. floridanum and C. crotalariae responded similarly to C. scoparium in many assays.     

Time and burial depth influencing the viability and bacterial colonization of sclerotia of Sclerotinia sclerotiorum

Sclerotia are the primary over wintering inoculum of Sclerotinia sclerotiorum (Lib.) de Bary. The effects of tillage on the primary inoculum are not well understood. The purpose of this research was to study sclerotial viability over time and between burial depths in soil, to identify bacteria colonizing and degrading the sclerotia, and determine whether these bacteria may be utilized as biological control agents. Correlation analysis indicated that a significant negative relationship existed between sclerotial viability and elapsed temporal factors (R²=−0.68, P<0.0001), and depth of burial (R²=−0.58, P<0.0001). After twelve months, sclerotia on the soil surface had the highest viability (57.5%), followed by those at the 5 cm depth (12.5%), and only 2.5% of those placed at the 10 cm depth remained viable. A significant negative relationship between sclerotial viability and bacterial populations also existed (R²=−0.60, P<0.0001). Two hundred and sixty-eight bacteria were isolated from sclerotia, 29 of which showed strong in vitro antagonism to the mycelial growth of S. sclerotiorum. Biodiversity of the inhibitory bacterial isolates was minimal on sclerotia from the soil surface and within all depths sampled at three months (i.e. in January). All burial depths within the April and July sampling dates produced bacterial diversities that were distinct from each other.     

Population and distribution of sclerotia of Rhizoctonia solani kühn in sugar beet field soil

The population and distribution of sclerotia of Rhizoctonia solani Kühn in two sugar beet field soils was determined at harvest by a sieving-flotation method. In rhizosphere soil (RS) and non-rhizosphere soil (NRS) from the most heavily infected roots of sugar beets, 1.43–2.5 and 0.83–1.0 sclerotia g^?1 dry soil were detected, respectively. In the soil around healthy sugar beet, these values were 0.04–0.12 and 0.03–0.04 sclerotia g^?1 dry soil. More sclerotia were always obtained from RS than from NRS. More than 80% of the sclerotia were in the upper 10 cm of soil and within 10 cm of diseased roots. Therefore, there is a non-uniform distribution of sclerotia of R. solani in soil.The sclerotial population in soil increased significantly with disease severity and a good correlation was obtained between the number of sclerotia and the disease severity on infected plants. Most of the sclerotia collected from the field soil ranged in size from 0.5 to 2.0 mm diameter.Viability of sclerotia increased as severity of crown rot increased and as the size of the sclerotia increased. Conversely, there was a progressive decrease in sclerotial germination with increasing depth in soil and increasing distance from the infected root.     

Reduction of sclerotial inoculum of Sclerotinia sclerotiorum with Coniothyrium minitans

Aspects of the biology of C. minitans and its potential for control of S. sclerotiorum were investigated.Temperatures below 7°C resulted in comparatively slow rates of germination and infection of sclerotia by C. minitans. The optimum temperature for germination, growth, infection of sclerotia, and destructive parasitism by C. minitans was 20°C. The optimum relative humidity for germination, growth and infection by C. minitans was above 95%.Autumn inoculations with suspensions of conidia, pycnidia and mycelium of C. minitans in the field resulted in negligible numbers of sclerotia remaining viable after 1 month. With culture-grown sclerotia 2 months were required for a similar reduction of sclerotial viability. In the absence of C. minitans mulching had no significant effect on sclerotial viability. In the presence of C. minitans mulching did, however, influence the viability and infection by C. minitans of culture-grown sclerotia. Populations of field sclerotia also differed from culture-grown sclerotia in that they harboured an internal population of microorganisms, which included C. minitans, and had a lower level of viability at the commencement of the treatments.A winter application of C. minitans did not result in significant infection of sclerotia nor in a reduction in viability of sclerotia. This failure is believed to have resulted from low temperatures and dry conditions.     

Survival of sclerotia of Sclerotium cepivorum berk. In muck soil as influenced by drying and the location of sclerotia in soil

The proportion of viable sclerotia of Sclerotium cepivorum placed in field plots in Burnaby, British Columbia, decreased with time (P = 0.05). Sclerotia that had been air-dried for 48–72 hr had a lower percentage survival than those that had not been dried. Sclerotia placed on the soil surface decayed more rapidly than those buried at 15 cm (P = 0.05). Loss of viability was due to decay of sclerotia rather than to a reduction in the ability of the sclerotia to germinate which did not decline with time (P = 0.05). After 16 months in the field 23.6; 2.1; 11.7 and 8.9% of the sclerotia remained viable in the not-dried buried, not-dried surface, dried buried and dried surface treatments respectively.     

Microbial populations involved in the suppression of Rhizoctonia solani AG1-1B by lignin incorporation in soil

Rhizoctonia solani causes worldwide losses in numerous crops. Sclerotia of R. solani remain viable for several years in soil and are an important source of primary infection. In this study the effect of soil incorporation of Kraft pine lignin, a side product of the paper industry, on viability of R. solani AG1-1B sclerotia was investigated. The efficacy of lignin was assessed in a sandy loam (Oppuurs) and a silt loam soil (Leest) collected from commercial fields in Belgium. Evaluating sclerotial viability after 4 weeks incubation in the two soils amended with 1% (w/w) Kraft pine lignin demonstrated a soil-dependent effect. In Leest soil the addition of lignin resulted in a significantly reduced sclerotial viability, together with an increased mycoparasitism by Trichoderma spp.; in Oppuurs soil, on the other hand, only a slight and insignificant reduction in sclerotial viability was observed. Based on phospholipid fatty acid analysis, different changes in microbial community structure upon lignin amendment were detected in the two soils. Both amended soils showed a significant increase in Gram negative bacteria. In Leest soil this increase was accompanied with a significantly higher increase in fungi and actinomycetes compared with Oppuurs soil. In addition, Kraft pine lignin resulted in both soils in a small but significant increase in manganese peroxidase activity and this increase tended to be higher in Leest soil. Manganese peroxidase produced by lignin-degrading basidiomycetes has previously been shown to degrade melanin, which protects the sclerotia against biotic and abiotic stress. We hypothesize that lignin-degrading fungi increased the susceptibility of the sclerotia to sclerotial antagonists such as Trichoderma, Gram negative bacteria and actinomycetes. Clearly, the effect observed here did not rely on the stimulation of one microbial group, but is the result of an interaction of different groups.     

Ecological and morphological characteristics of the sclerotia of Rhizoctonia solani Kühn produced in soil

Ecological and morphological characteristics of the sclerotia of R. solani produced in soil were compared among Japanese isolates belonging to five anastomosis groups (AG). Twenty-three out of 24 isolates produced sclerotia in soil and the number and size of sclerotia varied considerably among isolates within the same AG. Sclerotia from each group ranged in size from 0.25 mm to more than 2.5mm dia, with most between 0.5–1.0mm dia. Sclerotia formed in soil were smaller than those formed in pure culture. Sclerotia of each AG were tested for viability after 30–270 days incubation in artificially-inoculated soil. Sclerotia produced in soil were brown to dark-brown and these were divided into two distinct groups on external morphology: firm and irregularly globose to subglobose with either a pitted or smooth surface in one group, and rather soft with an indefinite shape in the other. On the basis of internal morphology, the sclerotia fell into three groups. Sclerotia of AG-1 were composed of three well-defined layers; the central cells had dense contents and were surrounded by an outer layer of empty cells which were bordered by a darkly-pigmented mucilaginous surface-layer. Sclerotia of AG-2, Type-1 had only two well-defined layers; The darkly-pigmented mucilaginous layer was absent. Sclerotia of the other AG were constructed of very loosely-arranged brown cells without any well-defined zones.     

Loss of nutrient-independence for germination by fungal propagules incubated on soils or on a model system imposing diffusive stress

Nutrient independent conidia of Cochliobolus victoriae and sclerotia of Sclerotium cepivorum, Macrophomina phaseolina, and Verticillium dahliae were incubated aseptically on sand through which a dilute salts solution percolated at a flow rate sufficient to inhibit germination. Propagules were then transferred to a static salts solution to assess germination. Conidia of C. victoriae and sclerotia of S cepivorum became nutrient-dependent (6% germination in salts solution) after 9 and 15 days on sand, respectively. Thirty-five days of diffusive stress were required to attenuate the nutrient-independence of M. phaseolina sclerotia. Sclerotia of V. dahliae lost little of their nutrient-independence even after 45 days of diffusive stress. Viability of C. victoriae and S. cepivorum was reduced after 45 days of diffusive stress, but viability of V. dahliae and M. phaseolina was not. Conidia of C. victoriae gradually became nutrient-dependent when incubated for several weeks on each of five soils. A loam and two sandy loam soils were more effective in decreasing the nutrient-independence of conidia than were two clay loams. Sclerotia of M. phaseolina also lost nutrient-independence when incubated on four of the five soils.Interruption of artificially imposed diffusive stress resulted in increased [¹⁴C]exudation from conidia of C. victoriae and sclerotia of M. phaseolina. Germinability on salts solution of C. victoriae conidia previously made nutrient-dependent was significantly increased, when the conidia were kept at 4°C for 3.5 days before germination assay. Conidia of C. victoriae made nutrient-dependent and then incubated on soils labeled with [¹⁴C]glucose, absorbed twice as much ¹⁴C from a loam and two sandy loam soils as from two clay loam soils. Following incubation on four of the five ¹⁴C-labeled soils, the germinability of the conidia in the absence of nutrients was significantly increased over that of conidia not incubated on these soils.The results suggest that a continued minimal stress may be needed to maintain the nutrient-dependence of some fungal propagules in soil. Interruption of nutrient stress appears to allow nutrient-dependent propagules an opportunity to recoup nutrients from the soil solution or to reorganize endogenous energy reserves whereby the potential for germination is increased.     

Diversity of potential microbial parasites colonizing sclerotia of Macrophomina phaseolina in soil

The colonization of Macrophomina phaseolina sclerotia by microbial parasites was evaluated in unsterilized field soil at different levels of soil moisture (0,-5, and-10 kPa) and temperature (20, 30, and 40°C). The maximum colonization of sclerotia was recorded in soil held at-5 or-10 kPa at 30–40°C. Trichoderma harzianum isolate 25–92 and Pseudomonas fluorescens isolate 4–92 were recorded as potential sclerotial parasites, and they significantly (P=0.05) reduced the germination of sclerotia by 60–63%. Cells of P. fluorescens and buffer-washed conidia of T. harzianum were completely agglutinated at 28°C with crude agglutinin of M. phaseolina. The ability of different antagonists to parasitize the sclerotia were correlated with the agglutination ability of the antagonists.     

An Improved Procedure for Determining Magnesium Fertilizer Dissolution in Field Soils

Abstract

The sequential extraction procedure currently used to measure magnesium (Mg) fertilizer dissolution in soils consists of removing dissolved Mg (step 1), and partially dissolved Mg (step 2), followed by an 18‐h extraction with 2 M HCl at room temperature to determine undissolved Mg (step 3). This procedure is satisfactory for soluble and moderately soluble Mg fertilizers but is not an accurate procedure for slightly soluble fertilizers, such as serpentine. When step 3 is replaced by a digestion procedure using 2 M HCl for 4 h at 90–95°C (improved step 3), the total serpentine Mg recovery (dissolved and undissolved Mg) from soil samples, either immediately after serpentine was added to soil or after a 21‐day incubation with moist soil, was about 100% compared to 40–50% by the original procedure. The improved procedure also increased the recovery of serpentine Mg applied to field soils. Therefore, this study recommends that the third step of the sequential extraction procedure be replaced by a 4 h digestion using 2 M HCl (90–95°C).     

Survival of Coniothyrium minitans associated with sclerotia of Sclerotinia sclerotiorum in soil

The development and survival of the mycoparasite Coniothyrium minitans associated with sclerotia of the plant pathogen Sclerotinia sclerotiorum was studied in pasteurised and non-sterile (untreated) soil. Using scanning electron microscopy, developing pycnidia were first seen within the sclerotial medulla at 7 days post-inoculation with the mycoparasite in pasteurised soil. However, by 14 days post-inoculation, pycnidia had developed fully in both pasteurised and non-pasteurised treatments, and conidial droplets were exuded onto the outer surface of the infected sclerotia. Thirty days post-inoculation, irrespective of soil treatment, the majority of the sclerotial medulla had been converted to pycnidia, with the sclerotial rind remaining largely intact. The pycnidia and dried intact droplets were still observed 6 months post-inoculation with C. minitans, although the conidia on the outer surface of the dried droplets had largely collapsed by this stage. Germinability studies at 10 months post-inoculation showed that approximately 13% of the conidia in dried droplets were still viable. This work shows the potential for infected sclerotia of S. sclerotiorum to provide a unique reservoir for the survival of C. minitans.     

Adenosine triphosphate (ATP) and microbial biomass in soil: Effects of storage at different temperatures and at different moisture levels

Abstract

On air‐drying, the ATP contents of two moist soils fell to about one quarter of their original values. When a freshly‐sampled soil (field temperature 5.5°C) was stored moist (43% water holding capacity) for 7 days at 25°C the ATP content increased from 4.54 to 7.84 μg ATP g^‐1 soil. Storage at 10°C caused a smaller increase; to 5.39 μg g^‐1 soil. Microbial biomass C also increased on storage but the relative increase was less than that of ATP. Thus the biomass C/ATP ratio fell from 234 in the freshly sampled soil to 168 in the soil stored moist for 7 days at 25°C. The ATP content declined to less than half its starting value if storage was under waterlogged conditions.

The ATP method for determining microbial biomass in soil depends on the use of a constant factor (5.85 mg ATP g^‐1 biomass C) for converting ATP content to biomass C. This factor came from work on soils that had been stored moist at 25°C for several days before biomass C and ATP measurements were made: it is only applicable to soils that have been stored in this way.     

Comparaison des aptitudes parasitaires de clones de Trichoderma vis-a-vis de quelques champignons a sclerotes

Nineteen monoconidial isolates (referred to as clones) of Trichoderma from different species aggregates, one isolate of Gliocladium virens, and one isolate of an Acrostalagmus sp. (that was naturally associated with sclerotia of Sclerotinia spp and Macrophomina phaseolina) were tested. They were incubated in controlled conditions, in sterile soil, with sclerotia of Corticium rolfsii, Sclerotinia minor, or S. sclerotiorum. At the end of appropriate periods of incubation (respectively 26, 20 and 8 days), the sclerotia were retrieved from soil and checked for invasion by the antagonist. Important differences between the parasitic ability of Trichoderma clones were noted. Clones from at least three different species (T. aureoviride, T. hamatum, T. harzianum) exhibited a high antagonistic activity. Activity of the G. virens isolate was at the same level as the best clones of Trichoderma, whereas no parasitic tendencies were found in the isolate of Acrostalagmus sp., thus confirming previous results.A rather good correlation was found between the capacity of the clones for attacking C. rolfsii sclerotia and their ability to parasitize both Sclerotinia.In conclusion, it is proposed that a screening with only one of the sclerotial species would give clones efficient against all three, and possibly against related sclerotial types.