Production and fungitoxic activity of Sch 642305, a secondary metabolite of <Emphasis Type="Italic">Penicillium canescens</Emphasis>

Production of fungitoxic extrolites was evaluated in culture filtrates of several isolates belonging to Penicillium canescens and P. janczewskii that showed some extent of inhibitory activity against the plant pathogenic fungus Rhizoctonia solani. In addition to griseofulvin and dechlorogriseofulvin that are already known in these species, curvulinic acid, previously unreported in Penicillium, was produced by all isolates assayed. Another extrolite recently characterized from a P. verrucosum strain by the name of Sch 642305 was detected in 5 isolates of P. canescens only. The purified compound completely inhibited mycelial growth of isolates of Rhizoctonia solani and other plant pathogenic fungi in␣vitro. The role of this extrolite as a possible biochemical determinant of antagonism toward plant pathogenic fungi, and implications concerning chemotaxonomy are discussed.     

Suppression of sugar beet damping-off caused by Rhizoctonia solani using bacterial and fungal antagonists

Rhizoctonia damping-off caused by Rhizoctonia solani Kühn, is one of the most damaging sugar beet diseases. It causes serious economic damage wherever sugar beets are grown. Biological control is an efficient and environmentally friendly way to prevent damping-off disease. Suppression of damping-off disease caused by R. solani was carried out by four isolates of Bacillus subtilis (Ehrenberg) Cohn as well as three isolates of each of Trichoderma harzianum Rifai and Trichoderma hamatum (Bonord.) Bainier. The effect of Bacillus and Trichoderma isolates against R. solani was investigated in vitro and tested on sugar beet plants under greenhouse conditions. Isolates of Bacillus and Trichoderma were able to inhibit the growth of R. solani in dual culture. Furthermore, Trichoderma isolates gave high antagonistic effect than isolates of B. subtilis. Under greenhouse conditions, coating seeds by T. harzianum and B. subtilis separately, reduced seedling damping-off significantly. However, applications of T. harzianum increased the percentage of surviving plants more than B. subtilis in comparison to control. The obtained results indicate that T. harzianum and B. subtilis are very effective biocontrol agents that offer potential benefit in sugar beet damping-off and should be harnessed for further biocontrol applications.     

Fungitoxicity of some higher plants againstRhizoctonia solani

Summary Leaves ofChenopodium ambrosioides exhibited strong fungitoxicity against the mycelial growth ofRhizoctonia solani causing damping off diseases of some seedlings. Minimum inhibitory concentration of the fungitoxic constituent isolated in form of essential oil, was found to be 1000 ppm at which it was fungicidal in nature. It exhibited broad range of antifungal activity and did not show any phytotoxicity on the germination and seedling growth ofPhaseolus aureus.     

Evaluation of the leaf juice of some higher plants for their toxicity against soil borne pathogens

Out of the leaf juices of eighteen plant species screened, only Eupatorium cannabinum exhibited complete toxicity against Pythium debaryanum, Fusarium oxysporum, Rhizoctonia solani and Sclerotium rolfsii. Shade drying of the leaves had no adverse effect, while oven drying produced an adverse effect on the fungitoxicity of the leaves of E. cannabinum. The crude leaf juice of E. cannabinum successfully inhibited damping-off (Fusarium oxysporum) infection of Pisum sativum seedlings.     

Disease cycle involved in damping-off of groundnut caused byRhizoctonia solani

Summary By taking into consideration of the results of various studies on damping-off of groundnut caused byRhizoctonia solani, the mechanisms leading to ultimate collapse and death of the seedlings, are described.     

The role of rhizosphere bacteria in herbicide-mediated increase in Rhizoctonia solani-induced cotton seedling damping-off

A study was carried out to determine the role of rhizosphere-residing bacteria in the observed pendimethalin- and prometryn-mediated increase in cotton seedling damping-off incidence caused by Rhizoctonia solani. Judging from the results, pendimethalin-mediated increase in disease incidence may be due to a decrease in the populations of indigenous cotton root colonizing bacteria in the presence of pendimethalin. Prometryn-mediated increase in disease may be due to a decrease in the populations of indigenous root colonizing bacteria as well as increased susceptibility of cotton seedlings to R. solani infection in the presence of prometryn. Pendimethalin and prometryn neither affected the growth of R. solani nor caused any visible change in seedling physical characteristics.     

Antagonism against Rhizoctonia solani and fungitoxic metabolite production by some Penicillium isolates

A number of Penicillium isolates were recovered in association to Rhizoctonia solani strains pathogenic on tobacco and from soil on plates pre-colonized by the pathogen itself. Their antagonism toward R. solaniAG-2-1 was evaluated in dual cultures in vitro. Inhibition of growth was evident to some extent in most pairings, while hyphal interactions referable to mycoparasitic relationships were not observed. However, the occurrence of plasmolysis and/or vacuolisation and the induction of monilioid cells were indicative of the release of bioactive compounds. Therefore, production of fungitoxic metabolites was tested by adding concentrated culture filtrates of each Penicillium isolate to the growth medium of R. solani. Complete and lasting inhibition was incited by culture filtrates of some isolates belonging to P. brevicompactum, P. expansum, and P. pinophilum. Three purified compounds, respectively mycophenolic acid, patulin and 3-O-methylfunicone, which were extracted from culture filtrates, were able to inhibit R. solani in vitro. Their production was also detected in dual cultures of the same Penicilliumstrains with R. solani prepared in sterilized soil and when the Penicilliumstrains were cultured directly on R. solani mycelium harvested from liquid cultures. The possible role of such metabolites in antagonism of the above-mentioned Penicilliumspecies against R. solani is discussed.     

Impact of two soil-applied herbicides on damping-off of cowpea caused byRhizoctonia solani

Summary Alachlor was more inhibitory toRhizoctonia solani growth than fluchloralin in potato dextrose broth (PDB). Infective capacity of the pathogen was not altered by growing it in a medium containing either of the herbicides. Cowpea seedlings grown in alachlor-treated soil were more susceptible toR. solani than those treated with fluchloralin and the untreated seedlings. Pre-sowing application of alachlor in soil (5 l a.i./kg) aggravated damping-off whereas fluchloralin decreased the disease incidence to nearly half of that in untreated soil in greenhouse pot tests (av. temperature 31±5°C). Both herbicides reduced damping-off in pots kept at constant temperature of 30°C and increased the disease incidence at 20°C. Fungus growth in culture was stimulated at 20° but was strongly inhibited at 30°C by both herbicides. Growth inhibition by herbicides was maximum in PDB of pH 8 and decreased steadily up to pH 5.Impact of fluchloralin and alachlor onR. solani damping-off of cowpea appears to be due to the predisposing effect by the herbicides on the susceptibility of the host and is influenced by atmospheric temperature.     

Grape marc compost: microbial studies and suppression of soil-borne mycosis in vegetable seedlings

Compost suppression of soil-borne diseases in horticultural crops has been attributed to the activities of antagonistic microorganisms. A great diversity of microorganisms, capable of suppressing pathogens naturally colonize compost. A large number of microbes appeared in microbiological analyses of grape marc compost. Most microorganisms were bacteria. Average percentages were 31% mesophilic and 28% thermophylic bacteria, 16% mesophilic actinomycetes and 20% thermophylic actinomycetes. Only a few mould and yeast morphologies were obtained, 4% and 1% respectively. Antagonist in vitro assays were performed with 432 microbial morphologies isolated from grape marc compost. The microbes isolated were extremely effective antagonists in in vitro assays against all the fungal pathogens tested. Seven microorganisms were selected for further bioassay with Rhizoctonia solani in radish, Fusarium oxysporum f. sp. radicis-cucumerinum in melon, and Phytophthora parasitica in tomato and two microorganisms with Pythium aphanidermatum in cucumber. Those experiments indicate that grape marc compost reduces the severity of Pythium damping-off in cucumber, but does not reduce the severity of Phytophthora root rot in tomato, Fusarium oxysporum f. sp. radicis-cucumerinum in melon and Rhizoctonia solani in radish. Better suppressive effects were not demonstrated by either compost or vermiculite amended with microbes selected from grape marc compost.     

Chemical,physical and biological control of carrot seedling diseases

In naturally infested soil containingPythium ultimum, P. acanthicum andPhytophthora megasperma, onlyP. ultimum was associated with root rot and damped-off seedlings. Damping-off was promoted by low soil temperatures and by flooding. Seedling stands were markedly reduced when seed was pre-incubated in soil at 12°C but not at 25°C or 35°C. Dusting carrot seed with metalaxyl significantly increased seedling stands in the field at rates from 1.5–6 g kg⁻¹ seed and in both flooded and unflooded, naturally infested soil at 3.15 g kg⁻¹. In greenhouse experiments using artifically infested soil,P. ultimum andP. paroecandrum caused damping-off of carrot seedlings andRhizoctonia solani reduced root and shoot weights.R. solani caused damping-off in nutrient-enriched soil.P. acanthicum andP. megasperma were not pathogenic to seedlings, although both fungi colonized roots. Soil populations of allPythium spp., particularlyP. ultimum, increased during growth of seedlings and population growth ofP. megasperma was promoted by periodic flooding. Infestation of soil withP. acanthicum did not reduce damping-off of carrot seedlings byP. ultimum orP. paroecandrum, but significantly increased root and shoot weights and decreased root colonization byR. solani P. acanthicum has potential as a biocontrol agent againstR. solani.     

林下参内生球毛壳菌FS-01对人参病原真菌的抑制作用

为探究林下参内生真菌球毛壳菌(Chaetomium globosum)FS-01菌株对人参病原菌的抑菌作用,该研究在实验室条件下,测定了FS-01菌株菌丝、发酵液和孢子悬浮液对人参黑斑病菌(Alternaria panax)、人参菌核病菌(Sclerotinia schinseng)、人参灰霉病菌(Botrytis cinerea)、人参立枯病菌(Rhizoctonia solani)、人参根腐病菌(Fusarium solani)5种人参病原菌的抑制作用。结果表明:内生真菌球毛壳菌FS-01对5种病原菌均有抑制作用,其中,对人参黑斑病菌的抑制作用最高,为30.80%,其次是人参立枯病菌、人参菌核病菌、人参根腐病菌和人参灰霉病菌; 发酵液抑菌实验结果表明,在加入内生真菌球毛壳菌FS-01菌株发酵液的PDA培养基上,对人参灰霉病菌的抑制作用最高,为82.09%,其次是人参菌核病菌、人参黑斑病菌、人参立枯病菌和人参根腐病菌; 孢子抑菌实验结果表明,在加入内生真菌球毛壳菌FS-01菌株孢子悬浮液的PDA培养基上,对人参黑斑病菌的抑制作用最高,为83.72%,其次是人参灰霉病菌、人参立枯病菌、人参菌核病菌和人参根腐病菌。综上结果认为,内生真菌球毛壳菌FS-01菌株对人参病原菌均有很高的抑菌作用,可作为人参病原菌的生防菌株资源。     

Variability in the Sensitivity of Rhizoctonia solani Anastomosis Groups to Fungicides

Tolclofos-methyl, iprodione and cyproconazole, among the eleven fungicides tested in vitro, gave consistently strong inhibition against all ten anastomosis groups (AGs) of Rhizoctonia solani. Carboxin, furmecyclox, thiabendazole, fenpropimorph and vinclozolin also inhibited all AGs but with wide variations in toxicity levels (EC90 values). Pencycuron showed strong activity against four AGs but was ineffective against the other six AGs. Generally, R. solani AGs were insensitive to fenarimol and imazalil. Tolclofos-methyl strongly inhibited 23 AG2-1 and 20 AG4 rapeseed/canola R. solani isolates from different locations in Saskatchewan, Alberta and Manitoba. The same isolates were also sensitive to iprodione, cyproconazole and carboxin. All AG4 canola isolates were insensitive to pencycuron (EC90 > 500 mg/l) while AG2-1 isolates showed highly variable levels of sensitivity with EC90s ranging from 0.5 to 220 mg/l. Tolclofos-methyl, applied to Brassica napus (canola) cv. Westar seed at 1 g a.i./kg, provided 75—100 % control of seedling damping-off in pots infested with AG2-1 or AG4 isolates. In parallel experiments, pencycuron (1 g a.i./kg seed) failed to control damping-off by AG4 canola isolates and gave variable disease control against AG2-1 isolates.     

Fungitoxicity of mineral oils against Rhizoctonia solani causing damping-off of mung bean (Phaseolus aureus)

Three mineral oils, BSO, EWOS and E9267 and one vegetable oil (mustard oil), did not appreciably inhibit the mycelial growth of Rhizoctonia solani. However, treatment of 100 g seeds of mung bean with 2 ml EWOS and E9267 oils controlled more than 90% of the pre- and post-emergence damping-off and protected seedlings in soil inoculated with R. solani 5 days after sowing. Soaking seeds in solutions of these oils or drenching the soil did not control damping-off. Mustard oil controlled only pre-emergence damping-off.     

Formulation of the Biocontrol FungusCladorrhinum foecundissimumto Reduce Damping-Off Diseases Caused byRhizoctonia solaniandPythium ultimum

Five isolates ofCladorrhinum foecundissimum, added to soilless mix as 10-day-old fresh bran preparations (1.0% w/w), significantly reduced (P≤ 0.05) damping-off of eggplant and pepper caused byRhizoctonia solanistrain R-23. After 4 weeks of growth, plant stands in the biocontrol-amended, pathogen-infested treatments (>80%) were comparable to those in the noninfested controls. Since plant stands were similar at 2 and 4 weeks, most of the disease was preemergence damping-off. The bran preparations also reduced saprophytic growth of the pathogen, and there was an inverse correlation (r²= −0.94) between saprophytic growth and eggplant stand. Added to soilless mix at a rate of 2.0% (w/w), alginate prill containing 20% fermentor-produced biomass of six biocontrol isolates ofC. foecundissimumreduced (P≤ 0.05) damping-off of eggplant caused byR. solani, but only the prill with biomass of isolates Cf-1 or Cf-2 yielded plant stands (>80%) comparable to that in the noninfested control. As with the bran preparations, there was also an inverse correlation (r²= −0.80) between saprophytic growth of R-23 and eggplant stand with the alginate prills. Alginate prill with biomass of Cf-1 or Cf-2 also reduced (P≤ 0.05) damping-off of eggplant and pepper caused by other isolates (195, NG-2, DPR-1) ofR. solani, but only the stands (>80%) of pepper were similar to that in the noninfested control. Alginate prill formulations ofC. foecundissimum(Cf-1, Cf-2, and Cf-3) also reduced (P≤ 0.05) populations of the pathogen and damping-off of eggplant and pepper caused byPythium ultimum(PuZ3). However, although the plant stands in the treatments were not as high as those in the noninfested controls, they were higher than those in the pathogen-infested controls. The treatments also reduced populations ofP. ultimumin the soilless mix so that there were inverse correlations between the pathogen population and eggplant stand (r²= −0.81) and pepper stand (r²= −0.78). Extruded flour/clay granules containing 5.0% biomass of Cf-1 and Cf-2, added toR. solani-infested soilless mix (2.0%), reduced (P≤ 0.05) damping-off of eggplant and pepper. However, only the Cf-2 treatments resulted in stands (>80%) equal to those in the noninfested controls for the crops after 4 weeks of growth. The influence of bran and alginate prill of Cf-1 or Cf-2 on the spatial spread ofR. solaniand its ability to incite damping-off of eggplant showed that prill with Cf-1 or Cf-2 and bran with Cf-2 were equally effective in reducing the spread of the pathogen from the point source of the inoculum to the center of the flats.     

Formulation of a Streptomyces Biocontrol Agent for the Suppression of Rhizoctonia Damping-off in Tomato Transplants

Formulations of a Streptomyces biological control agent for Rhizoctonia damping-off in tomato seedlings were developed for the first time from vegetative propagules obtained from actively growing, nonsporulating liquid cultures. Alginate beads, durum flour (starch) granules, and talcum powder formulation of this new actinomycetous antagonist (Streptomyces sp. Di-944) isolated from the rhizosphere of field-grown tomato (Lycopersicon esculentum) suppressed damping-off caused by Rhizoctonia solani in tomato plug transplants (cv. Bonny Best) in a peat-based, soilless potting mix under greenhouse conditions. For formulations, vegetative biomass of Streptomyces sp. Di-944 from 3-day-old liquid fermentation in yeast extract–malt extract–glucose broth was lyophilized and pulverized to obtain fragments of viable vegetative filaments. The pulverized biomass had an initial viable count of 2 × 10⁷colony forming units/g and retained 100% viability for 2 weeks when stored at 4°C. Formulations stored at 4°C had a longer shelf life than those stored at 24°C based on viability at 2-week intervals over a 6-month storage period. In addition, dual culture tests showed declining efficacy for surviving Streptomyces propagules in formulations during this storage period. At 4°C, the powder and granular formulations were found to be the most stable and were shown to be 100% viable after 14 and 10 weeks of storage, respectively. However, at the end of 24 weeks, the number of viable propagules in the powder and granular formulations declined to 1.2 × 10⁵ and 7 × 10³ colony forming units/g, respectively. Alginate beads were the least stable in storage. Even at 4°C, 6.9 × 10⁴ and 7.3 × 10² viable propagules/g formulation were detected at the end of 12 and 24 weeks, respectively. The talcum powder formulation delivered to tomato seeds as a seed-coating was the most effective biocontrol treatment. It suppressed damping-off in 10-day-old tomato transplants by almost 90% compared to 30 and 22% damping-off reduction when alginate beads or starch granules were delivered concomitantly with tomato seeds. Seed-coating with powder formulation of the biocontrol agent was as effective as drench application of the fungicide, oxine benzoate (No-Damp), in controlling Rhizoctonia damping-off and superior to the commercial biocontrol agent, Streptomyces griseoviridis (Mycostop), applied to tomato seeds as seed-coating.     

A quantitative bioassay for extracellular metabolites that antagonize growth of filamentous fungi,and its use with biocontrol fungi

A bioassay and an empirically derived formula were developed to quantify fungitoxic effects. This bioassay can be easily performed and objectively read, and it is suitable for low-volume samples originating from aqueous or organic solvents. The formula defines the Inhibition Index (I), a single value that incorporates both the response to concentrations of the inhibitory compound and the persistence of inhibition. Antagonistic efficacy of metabolites produced by biocontrol strains of Trichoderma spp. were measured based on inhibition of growth of Rhizoctonia solani. Although the bioassay itself was not influenced by pH or light conditions, these factors affected metabolite production or activity. Aqueous extracts from light-grown germlings of Trichoderma virens inhibited R. solani more than extracts from germlings grown in the dark. Low pH increased the inhibitory activity of extracts from T. virens. Tests of fungal strains developed for biocontrol demonstrated that the bioassay reflected their activity both in the field and in other in vitro tests. The bioassay and formula are readily adapted for use with other fungi.This revised version was published online in October 2005 with corrections to the Cover Date.     

Rhizotron studies on Zea mays L. to evaluate biocontrol activity of Bacillus subtilis

The effect of Bacillus as a biocontrol agent against some root-rot fungi was tested using maize (Zea mays L.) in rhizotrons placed in a growth chamber with relative humidity 60% with a 12 h photoperiod and day and night temperatures of 24 and 18°C respectively. Rhizoctonia solani Kühn caused pre-emergence damping-off in the untreated maize seeds showing weak and soft roots as observed through the perspex rhizotrons. Image analysis was used to quantify the effects of Bacillus treatment on seedlings infected with Pythium sp. Bacillus B77 and B81 were most effective in the control of the pathogen, R. solani which achieved a biocontrol activity of 24 and 35% respectively with regard to shoot dry biomass while B81 achieved 48% biocontrol with reference to root dry biomass. There was no effect on the root area. For root dry biomass, B81, B69, B11 and B77 showed higher biocontrol activity in comparison to the control. Pythium sp. caused pre- and post emergence damping- off in the untreated seeds. Root rot of the maize seedlings caused by Pythium sp. was slightly controlled by Bacillus B69 and B81 which achieved biocontrol activity of 18 and 11% respectively. For the biocontrol of Fusarium solani, Bacillus B77, B69, B81 achieved biocontrol activity of 50, 48 and 33% respectively with reference to root dry biomass.     

Biological Control of Phoma and Pythium Damping-Off of Sugar-Beet with Pythium oligandrum

Flooding freshly harvested oospores in sterile distilled water (SDW) for several days enhanced germination in 3 out of 4 isolates of Phythium oligandrum. Treatment of SDW-flooded oospores with myo-inositol increased germinability during the first 20 days of storage at 15°C. Seed dressing with oospores of P. oligandrum controlled pre- and post-emergence damping-off of sugar-beet caused by soil-borne P. ultimum and seed-borne Phoma betae. For some isolates, flooded oospores in SDW and treatment with myo-inositol increased efficacy of the seed dressing. However, no significant control of damping-off caused by Rhizoctonia solani was observed. On corn-meal agar, P. oligandrum coiled around and penetrated hyphae of P. ultimum and R. solani, but did not interfere with Ph. betae.     

Variations in the cultural characteristics of Rhizoctonia solani,and its antagonists: Aspergillus terreus and Aspergillus flavus occurring in the rhizosphere of cotton

Summary Cultural studies onRhizoctonia solani, the causal agent of damping-off of cotton, as well as on two fungi of its antagonistic rhizospheric microflora, namelyAspergillus terreus andAspergillus flavus have shown coincidence of some of their cultural characteristics. However,R. solani produced mycelial growth far ahead both antagonists except at 37° C and at pH 4, at its optimum temperature. It is expected that for a successful biological control ofR. solani in the soil,A. terreus is applied at a soil-temperatured above 35° C in an acid medium.     

Cloning of Genes Involved in the Synthesis of Pyrrolnitrin from Pseudomonas fluorescens and Role of Pyrrolnitrin Synthesis in Biological Control of Plant Disease

A soil isolate of Pseudomonas fluorescens (BL915) was shown to be an effective antagonist of Rhizoctonia solani-induced damping-off of cotton. Investigation of the biological basis of this antagonism revealed that the strain produces pyrrolnitrin, a secondary metabolite known to inhibit R. solani and other fungi. Mutants of strain BL915 that did not produce pyrrolnitrin and did not suppress damping-off of cotton by R. solani were generated by exposure to N-methyl-N′ -nitro-N-nitrosoguanidine. A gene region that was capable of restoring pyrrolnitrin production to the non-pyrrolnitrin-producing mutants and of conferring this ability upon two other P. fluorescens strains not otherwise known to produce this compound or to be capable of suppressing damping-off caused by R. solani was isolated from strain BL915. The non-pyrrolnitrin-producing strains (mutants of BL915 and the other two P. fluorescens strains) which synthesized pyrrolnitrin after the introduction of the gene region from strain BL915 were also shown to be equal to strain BL915 in their ability to suppress R. solani-induced damping-off of cotton. These results indicate that we have isolated from P. fluorescens BL915 a gene(s) that has a role in the synthesis of pyrrolnitrin and that the production of this compound has a role in the ability of this strain to control damping-off of cotton by R. solani.