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1.
Ten isolates of Trichoderma spp were examined for their ability to antagonize growth and to parasitize mycelium of Sclerotium rolfsii (Sr-1) on agar media, to inhibit germination of sclerotia of S. rolfsii on natural soil plates and to sporulate on the sclerotia, and to protect bean seedlings against the pathogen in the greenhouse. A high negative correlation (r = ?0.844) was observed between plant stand in the greenhouse and sclerotial germination on soil plates but not with antagonism on agar plates. Three isolates of T. harzianum (Th-7, Th-20, WT-6) and one of T. hamatum (TRI-4) were especially effective in reducing sclerotial germination and controlling disease in the greenhouse. Three isolates of Trichoderma spp (WT-6, TMP, and TRI-4), effective in reducing sclerotial germination of isolate Sr-1, also prevented sclerotial germination in four out of five additional S. rolfsii isolates studied.  相似文献   

2.
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.  相似文献   

3.
This paper describes the identification and utilisation of a sequence-characterised amplified region (SCAR) marker specific for the Trichoderma virens biocontrol isolate GV4. The marker was developed from a RAPD-PCR amplification product unique to isolate GV4. When used as a hybridisation probe in Southern blot analysis, it hybridised to the DNA of the species T. virens alone and not to that of other Trichoderma species or closely related genera Gliocladium and Verticillium. The marker also produced a GV4-specific RFLP, distinguishing it from other T. virens isolates when probed to blots with HindII, BamHI or PstI genomic DNA digests. Primers designed from the sequence of the RAPD marker produced a diagnostic amplification product of 346 bp for GV4 alone, distinguishing it from all other test isolates. With the exception of one, test isolates did not produce an amplification product with the SCAR primers. The exception was a single Verticillium psalliotae isolate (ICMP5509) that produced a product of approx. 400 bp that was easily distinguished from the 346 bp product of GV4. The reliability of the SCAR-based diagnostic test was further improved with the introduction of a positive PCR reaction control to each test, achieved by converting the test to a duplex PCR system. Two universal primers flanking the two ITS and the 5.8S region of the ribosomal gene complex were introduced to each reaction to provide a test for PCR reaction inhibitors to eliminate false negatives in the diagnosis. Amplification of this multi-copy genomic region did not reduce diagnostic sensitivity of the single copy SCAR marker. To further increase the sensitivity of detecting GV4 propagules while maintaining a fast sample assessment assay, soil was amended with cornmeal, as a nutrient source, and a mix of antibiotics to favour Trichoderma growth. The soil mix was subsequently incubated for 5 d before total DNA was extracted. Under these conditions, the duplex soil PCR assay detected GV4 down to a concentration of 10 spores g−1 soil in non-sterile agricultural field soil. This study is the first to report the use of a duplex-PCR diagnostic bioassay for a species within the Hypocrea/Trichoderma genus.  相似文献   

4.
Some members of the fungal genus Trichoderma are able to colonize and destroy sclerotia, the thick-walled resting structures of the soilborne plant pathogenic fungus Sclerotinia sclerotiorum, thus providing a potential means of biological disease control. However, current methods to detect and quantify colonization of sclerotia are labor-intensive, and generally qualitative rather than quantitative in nature. Our objective was to develop quantitative real-time PCR (polymerase chain reaction) methods to detect and measure colonization of sclerotia by Trichoderma spp. Specific PCR primer/probe sets were developed for Trichoderma spp. and for S. sclerotiorum. A total of 180 ITS1 (internal transcribed spacer) and ITS2 sequences from different species in the genus Trichoderma were aligned, and consensus sequences were determined. Six candidate primer sets were based on conserved regions of the consensus sequence, and the specificity of each nucleotide sequence was examined using BLAST (Basic Local Alignment Search Tool; NCBI) software. Each candidate primer set was tested on genomic DNA of T. harzianum strain ThzID1-M3, as well as six different Trichoderma isolates from soil, and on genomic DNA of S. sclerotiorum. The optimum primer/probe set selected, TGP4, successfully amplified genomic DNA of all Trichoderma isolates tested, and showed high precision and reproducibility over a linear range of eight orders of magnitude, from 85 ng to 8.5 fg of T. harzianum genomic DNA. TGP4 did not amplify S. sclerotiorum DNA. A specific PCR primer/probe set (TMSCL2) was developed for S. sclerotiorum, based on the calmodulin gene sequence. TMSCL2 did not amplify Trichoderma DNA. Quantitative real-time PCR with the primers then was evaluated in experiments to test differential effects of two soil moisture levels (−50 kPa, −1000 kPa matric potential) on biocontrol of S. sclerotiorum by indigenous Trichoderma spp. Periodically over 40 days, Trichoderma and S. sclerotiorum DNA in sclerotia were quantified by PCR with appropriate primers. Over 90% of the sclerotia were colonized by indigenous Trichoderma spp. at −1000 kPa, over the 40-day period, compared to only 60% at −50 kPa. In addition to determining incidence of colonization, the PCR method allowed measurement of the extent of sclerotial colonization, which also was significantly greater in the drier soil. Quantitative real-time PCR with the TGP4 primer/probe set provides a sensitive detection and measurement tool to evaluate colonization of sclerotia by Trichoderma spp.  相似文献   

5.
The ability of Trichoderma harzianum isolate 203 to attack the soil-borne plant pathogen Sclerotium rolfsii is apparently connected with the production by the isolates of chitinase and β-(1,3)-glucanase inside the attacked sclerotia during parasitism.SEM and TEM micrographs show that the mycoparasite degraded walls of sclerotial cells and the attacked cells lost their cytoplasmic contents. It is assumed that T. harzianum utilizes sclerotial cell contents thus enabling it to sporulate intensively on the sclerotial surface and inside the digested cells.  相似文献   

6.
《Applied soil ecology》2007,35(1):21-24
Field studies were conducted over two seasons to investigate effects of random versus highly aggregated spatial arrangements of sclerotia of S. sclerotiorum, and effects of biocontrol agent density and formulation additives, on colonization of sclerotia by Trichoderma spp. Application of T. harzianum encapsulated in alginate pellets with either bran or polyethylene glycol additives increased the percentages of sclerotia colonized in both years, but there was no difference between additives in either year. Higher pellet densities (200 pellets/m2 versus 40 pellets/m2) resulted in higher proportions of sclerotia colonized by Trichoderma spp. in one season but not in the other. However, when sclerotia were in highly aggregated spatial patterns, significantly higher percentages were colonized in both years, compared to sclerotia in random distributions.  相似文献   

7.
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.  相似文献   

8.
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.  相似文献   

9.
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.  相似文献   

10.
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.  相似文献   

11.
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.  相似文献   

12.
Factors affecting longevity of sclerotia in sandy clay loam (s.c.l.) and sandy loam (s.l.) were examined, using sclerotia from a laboratory culture of S. sclerotiorum and from natural infestations on beans and lettuce.Survival of sclerotia from culture and lettuce was compared in s.l. Recovery and viability were less, and incidence of Fusarium, Mucor and Trichoderma spp. greater, in sclerotia from lettuce than from culture. Rinds of sclerotia from lettuce were more perforated than those from culture.Burial of sclerotia at 4 cm for 35 weeks reduced recovery of sclerotia to zero in s.c.l. and by 50% in s.l. At the soil surface recovery was reduced by 55% in s.c.l. and by 10% in s.l. Less than 50% of sclerotia recovered were viable. Neither a chloropicrin-methyl bromide fumigant nor a tomato compost treatment affected recovery or viability. Fumigation increased incidence of Trichoderma spp. and decreased incidence of Fusurium and Mucor spp. isolated from sclerotia.Apothecia were produced over 6 weeks in s.c.l. and over 20 weeks in s.l. Production was increased by the low rate of fumigant in s.c.l. and by tomato compost in s.l.  相似文献   

13.
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.  相似文献   

14.
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.  相似文献   

15.
《Applied soil ecology》2007,35(1):237-246
Effectiveness of Trichoderma strains for biocontrol of soilborne pathogens requires an improved understanding of soil and root ecology of this fungus. We compared the population dynamics of Trichoderma hamatum strain T382 (T382) and indigenous Trichoderma spp. in soil and on roots in different strawberry production systems. Strawberry transplants, either amended or not-amended with Trichoderma biocontrol strains, were planted in field soil left untreated or treated with soil fumigant, compost, and compost-amended with T382. Soil and root samples were taken between October and June of two production seasons (2002-03 and 2003-04), and Trichoderma populations were assessed by plating soil dilutions and root pieces onto selective medium. Identity of T382 was confirmed using strain-specific primers. T382 became established and maintained a stable population of 103 cfu/g soil throughout the growing season when added to field soil in amended compost, but T382 was rarely isolated from strawberry roots. Populations of indigenous Trichoderma spp. were up to 60-fold greater in fumigated soil than in any other soil treatment. Indigenous Trichoderma spp. were isolated from a greater proportion (20–50%) of roots in fumigated soil than from roots in the other treatments (0–20%). Transplant treatments did not significantly affect Trichoderma populations on roots or in soil during field production. This study showed that compost may be used as a substrate to establish and promote survival of Trichoderma in field soil, and illustrates how soil manipulation can affect population dynamics of indigenous Trichoderma spp. on roots and in soil.  相似文献   

16.
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.  相似文献   

17.
Sclerotinia stem rot is one of the most serious diseases in rapeseed (Brassica napus) due to the lack of resistance sources. A high level of resistance was reported in Brassica oleracea cytodeme, one of parental species of rapeseed. In this study, a panel of 55 resynthesized lines of B. napus (RS lines) derived from seven wild and two cultivated types of B. oleracea was evaluated for Sclerotinia resistance over 2 years. Relative to ‘Zhongyou 821’, a cultivar of B. napus with partial resistance against S. sclerotiorum, RS lines exhibited stronger stem resistance. Although the resistant level of RS lines was lower than that of corresponding parental B. oleracea, a moderate correlation between resistance of RS line and corresponding parental B. oleracea type was found both for leaf (r = 0.74, P = 0.02) and stem (r = 0.69, P = 0.04). Our data suggests that the RS lines are important resources to improve Sclerotinia resistance of current rapeseed. A breeding strategy is discussed to enhance the Sclerotinia resistance of rapeseed by using B. oleracea.  相似文献   

18.
Relationship between the biocontrol fungus Trichoderma harzianum ThzID1-M3 and the plant pathogen Fusarium solani f.sp. pisi (Fsp) was determined in this study. Dual culture assay indicated the competitive interaction between ThzID1-M3 and Fsp. Alginate pellets of ThzID1-M3 and Fsp conidia were added to non-sterile soil with pea seeds. The interaction between Trichoderma and Fsp adversely affected their establishment in soil. The addition of ThzID1-M3 significantly reduced the Fsp population, as well as the colonization of roots by Fsp. In contrast, the added Fsp significantly reduced the proliferation of ThzID1-M3 and Trichoderma spp. The results suggest that the competition may be the main mechanism of the antagonistic activity of Trichoderma against Fsp.  相似文献   

19.
At least 75% of the sclerotia of Macrophomina phaseolina survived for 1 yr in most natural soils kept at 26°C and at 50–55% 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 1 yr. Soil pH had very little or no effect on sclerotial survivability. Of three organic amendments tested (alfalfa hay, chitin, pine needles) only ground alfalfa hay at 0.8% (w/w) reduced survivability of sclerotia in soil by about 75% in a year. Alfalfa hay at 0.4% reduced survivability by 36%. Various N sources added at 200 μg Ng?1 soil had no effect on survival. Of 13 fungicides tested, only benomyl and captan at 20 μg a.i. g?1 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.h.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 (50–55% 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 80–90% in moist soil (50–55% 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 1-week freezing (?5°C).  相似文献   

20.
The effects of forest fires on the soil mycotlora were investigated in a Pinus contorta forest in Alberta, where it was found that species of Trichoderma and Penicillium were reduced in the burned plot, whereas Gelasinospora sp occurred only in the burned plot: Cylindrocarpon destructans appeared not to be affected by fire.The response of fungi to aqueous extracts of burned and unburned litter, measured as linear growth on agar, showed that, of the isolates tested, all but C. destructans were inhibited by the burned litter extract; C. destructans grew better on the burned litter extract. An examination of spore germination rates and growth in liquid culture showed that Trichoderma polysporum and Penicillium janthinellum were both inhibited by burned litter extracts whereas C. destructans was not. Gelasinospora sp did not grow in liquid culture, nor did it produce spores after being kept in culture for some time.It was concluded that species of Trichoderma and Penicillium were killed by the heat of the fire, and subsequently unable to rccolonize the upper layers of the soil, due to an inhibition of spore germination and growth by the chemical products of burning. C. destructans on the other hand may have been able to recolonize quickly as it appeared to be stimulated in its linear growth rate by the chemical products of burning, and its spore germination rate was only marginally lowered. The occurrence of Gelasinospora sp following fire is possibly explained by its extremely rapid growth rate, and the possibility of its ascospores being more able to withstand high temperatures in the soil.In the light of recent reports, indicating that some species of Trichoderma and Penicillium are actively antagonistic to other fungi, it is suggested that their absence after fire, in the area studied, may permit a high inoculum of C. destructans to develop in the soil, which could possibly result in a high incidence of disease in developing pine seedlings  相似文献   

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