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.     

Formulation of a soil biofungicide for control of damping-off of Chinese cabbage (Brassica chinensis) caused by Rhizoctonia solani

A granulate biofungicide named PBGG was developed by combining Pseudomonas boreopolis with Brassica seed pomace, glycerin and sodium alginate. Application of 1.0% (w/w) of PBGG to the soil infested with Rhizoctonia solani AG-4 significantly reduced the percentage of colonization of cabbage seeds by the pathogen and stimulated proliferations of actinomycetes. Among Streptomyces populations, Streptomyces padanus (strain SS-07) and Streptomyces xantholiticus (strain SS-09) were the two most effective strains with strong antagonistic effects to R. solani. Application of 1% (w/w) PBGG in soil significantly increased population densities of S. padanus and S. xantholiticus. Treating soil with S. padanus (strain SS-07) or S. xantholiticus (strain SS-09) alone or in combination with 1% (w/w) PBGG significantly reduced the percentage of colonization of cabbage seeds by R. solani, compared to untreated controls. S. padanus was the most effective agent in reducing the percentage of seed colonization by the pathogen. Results of greenhouse tests showed that the most effective treatment was the amendment of pathogen-infested soil by S. padanus + 1% (w/w) PBGG which resulted in a disease incidence of 6.5–8.6%, compared to 27.8–31.7% for the treatment of S. xantholiticus + 1% (w/w) PBGG, 36.9–38.6% for the treatment of 1% (w/w) PBGG alone, and 61.8–64.8% for the treatment of control (unamended soil). Results of greenhouse and field tests also showed that soil amendment of 1% (w/w) PBGG alone or 1% (w/w) PBGG mixed with S. padanus (strain SS-07) or S. xantholiticus (strain SS-09) was not harmful to seed germination of Chinese cabbage. The study suggests that amendment of R. solani infested soil with S. padanus + 1% (w/w) PBGG prior to sowing is an effective method for control of damping-off of Chinese cabbage.     

Control of pre- and post-emergence damping-off of potato seedlings caused by Rhizoctonia solani in Peru

Fungicide treatment of soil and true potato seed and the use of subsoil as a growth medium significantly reduced pre-emergence damping-off caused by Rhizoctonia solani. Attempts were made to determine safe levels of fungicides for soil and seed treatments. For seed, safe levels varied between 0–9% a.i. for pentachloronitrobenzene (PCNB) and 3-0% a.i. for mancozeb (dithane M-45). Fungicides only marginally reduced post-emergence damping-off of transplants raised from true potato seed but gave significant increase in yield. The prospects of using fungicide treatment of true seed to control damping-off in seed beds and in direct field sowing are discussed.     

Co-utilization of Bacillus subtilis and Flutolanil in controlling damping-off of tomato caused by Rhizoctonia solani

Damping-off of tomato caused by Rhizoctonia solani was controlled in a pot test using the biological agent, Bacillus subtilis RB14-C, and the chemical pesticide, Flutolanil. The co-utilization of B. subtilis RB14-C, and Flutolanil decreased the amount of Flutolanil used from 375 g/pot when Flutolanil was used alone to 94 g/pot, while exerting the same effect of reducing disease occurrence.     

Fungicidal control of damping-off and seedling root rot in Brassica species caused by Rhizoctonia solani in the growth chamber*

Eight fungicides (benodanil, carboxin, cyproconazole, fenpropimorph, fur-mecyclox, iprodione, pencycuron and tolclofos-methyl) were evaluated, under growth chamber conditions, as seed treatments against pre-emergence damping-off and post-emergence seedling root rot in six Brassica species. Five cultivars of B. rapa, four cultivars of B. juncea, four cultivars of B. napus and one cultivar/ strain from each of B. carinata, B. nigra and B. oleracea were grown in soilless mix infested with an isolate of Rhizoctonia solani AG-2-1. B. nigra and B. juncea were considerably less susceptible to R. solani than the four other species. Cyproconazole at 0.05-0.1 g a.i./kg seed and the other fungicides at 2–4 g a.i./ kg seed provided almost complete control of pre-emergence damping-off in most Brassica species and their cultivars. Their efficacy varied against the post-emergence seedling root rot. Furmecyclox, iprodione, tolclofos-methyl and pencycuron consistently gave good control of seedling root rot in all Brassica species and their cultivars. Benodanil and fenpropimorph provided moderate control, and carboxin and cyproconazole gave poor control against root rot. Efficacy of carboxin, cyproconazole, benodanil and fenopropimorph against seedling root rot varied significantly (P ≤ 0.05) among cultivars within a Brassica species.     

Isolation and characterization of a novel Burkholderia cepacia with strong antifungal activity against Rhizoctonia solani

Strain CF-66 with strong antifungal activity against Rhizoctonia solani was isolated from compost samples. It is clearly demonstrated that strain CF-66 is belonging to Burkholderia cepacia complex by the morphological and biochemical tests and 16S rDNA sequence. The B. cepacia complex consists of a group of bacteria currently organized into nine genomovars, among them genomovar II and genomovar III, contain the highly epidemic strains. However, it was known that strain CF-66 is not a member of genomovar II or III of the B. cepacia complex by species-specific polymerase chain reaction assay. In this study, the antifungal compound CF66I produced by strain CF-66 was purified and characterized. Based on the nuclear magnetic resonance, GC-MS spectral and infrared spectral data, CF66I was confirmed to have amide bonds, α-metyl fatty acid, bromine, and some structural units such as CH₂CH₂O. CF66I is stable to high temperature, proteolytic enzymes, and organic solvents. CF66I inhibit the growth of a variety of plant pathogenic fungi and pathogenic yeast, whereas bacterial cells are unaffected. CF66I mainly reduced hyphal extension rates in a dose-dependent manner and induced severe change in cell morphology that resulted in swelled and formed very short hyphae with multiple branches.     

Etiology and control of durian foliar blight and dieback caused by Rhizoctonia solani

Foliar blight and dieback of durian seedlings and trees in Peninsular Malaysia was found to be caused by Rhizoctonia solani (teleomorph - Thanatephoms cucumeris) The fungus grew well and produced abundant sclerotia at temperatures higher than 24°C with an optimum at 28°C. It grew poorly at 35°C and did not grow at 10°C. The strains studied were found to belong to the anastomosis group AG-1. They were pathogenic on durian, papaya, cucumber, long bean, Mikania weed, padi, musk melon, mung bean, Zoysia grass, Bermuda grass, and St Augustine grass. They were mildly pathogenic on groundnut, and non-pathogenic on maize, guava and Brassica‘pak choy’. The disease was effectively controlled by foliar sprays of pencycuron and benomyl; triadimefon and an antagonistic bacterium suspension treatment were less effective and quintozene-etridiazole mixture gave poor disease control.     

Biocontrol of Rhizoctonia Damping-off of Tomato by Bacillus subtilis Combined with Burkholderia cepacia

Bacillus subtilis strain RB14‐C and Burkholderia cepacia strain BY were used in combination to control damping‐off of tomato plants caused by Rhizoctonia solani. Microcosm tests showed complete inhibition of R. solani growth on filter disks buried in soil added with the mixture of both bacteria. Single BY inhibited the fungus, but not completely, and RB14‐C had only slight inhibitory effect on pathogen growth. The efficacy of this combining treatment was checked in pot experiments, where bacteria were applied to the soil in several combinations: RB14‐C and BY together 4 days before seed planting, RB14‐C 4 days and BY 2 days before seed planting, RB14‐C 4 days and BY immediately before seeds. The effect of these treatments on population of R. solani in soil and infection of plants was compared with the activity of single application of each agent. All bacterial treatments significantly decreased damping‐off of tomato plants. The best control was obtained when BY was added 2 days after RB14‐C. In this treatment plant protection was significantly higher than that obtained in other combined applications and obtained by single strains, except BY added to the soil 4 days before seed planting. The lowest suppression indicated BY introduced to the soil before seed planting. RB14‐C only slightly decreased number of R. solani in the soil. In contrast, BY drastically reduced population of the pathogen. However, there was not a clear relation between decrease of pathogen density in soil and the rate of plant infection. The results show that combination of B. subtilis RB14‐C with B. cepacia BY can lead to greater damping‐off suppression than biocontrol exhibited by these strains used separately, but the effect of combining bacterial agents was clearly related to the order in which both agents were introduced.     

Quantification of rice sheath blight progression caused by Rhizoctonia solani

Rhizoctonia solani has a wide host range, including almost all cultivated crops and its subgroup anastomosis group (AG)-1 IA causes sheath blight in rice. An accurate measurement of pathogen’s biomass is a convincing tool for enumeration of this disease. Mycological characteristics and molecular diagnosis simultaneously supported that all six strains in this study were R. solani AG-1 IA. Heterokaryons between strains Rs40104, Rs40105, and Rs45811 were stable and viable, whereas Rs40103 and Rs40106 did not form viable fused cells, except for the combination of Rs40106 and Rs40104. A primer pair was highly specific to RsAROM gene of R. solani strains and the amplified fragment exists as double copies within fungal genome. The relationship between crossing point (CP) values and the amount of fungal DNA was reliable (R ² >0.99). Based on these results, we determined R. solani’s proliferation within infected stems through real time PCR using a primer pair and a Taqman probe specific to the RsAROM gene. The amount of fungal DNA within the 250 ng of tissue DNA from rice cv. Dongjin infected with Rs40104, Rs40105, and Rs45811 were 7.436, 5.830, and 5.085 ng, respectively. In contrast, the fungal DNAs within the stems inoculated with Rs40103 and Rs40106 were 0.091 and 0.842 ng. The sheath blight symptom progression approximately coincided with the amount of fungal DNA within the symptoms. In summary, our quantitative evaluation method provided reliable and objective results reflecting the amount of fungal biomass within the infected tissues and would be useful for evaluation of resistance germplasm or fungicides and estimation of inoculum potential.     

Identification of anastomosis group of Rhizoctonia solani, the causal agent of seed rot and damping-off of bean in Iran

Bean is one of the major crops in Iran. Seed rot and damping-off caused by Rhizoctonia solani is the most important disease of bean. In this research, infected roots and seedlings of beans were collected from different fields of Tehran Province. The samples were sterilized with 10% sodium hypochloride (5% stock) and incubated on PDA surface in petri-dishes. The purified fungi kept on filter paper and identified, pathogenicity test of R. solani was carried out on 2 cultivars of bean (red bean cv. Naz and white bean cv. Dehghan) and it determined. For identification of the anastomosis groups, the discs of cultured media with 5 mm. diameter of standard AG placed on one side of microscopic slides covered with water agar (2%) of 1 mm. thick and the isolates of the fungus on another side of slide about 2 cm away from each other. Experiment carried out in 4 replications. The cultures were incubated in 25 +/- 1 degrees C incubator for 24 hours, then the mycelial contact stained with lactophenol, cotton blue and hyphal anastomosis looked for under the light microscope with 10 x 40 and 10 x 100 magnifications. As a result, anastomosis groups: AG4, AG4HGII, AG2-2-2B and AG6 determined, frequency of these groups were 64, 18, 2, 16%, respectively. The group AG6 and subgroups AG4HGII and AG2-2-2B are introduced as new anastomosis groups on bean in Iran.     

Biocontrol of Rhizoctonia solani damping-off disease in cucumber with Bacillus pumilus SQR-N43

Biological control is an efficient and environmentally friendly way to prevent damping-off disease. Micrographs were used to investigate the ability of Bacillus pumilus (B. pumilus) SQR-N43 to control Rhizoctonia solani (R. solani) Q1 in cucumbers. The root colonization ability of B. pumilus SQR-N43 was analyzed in vivo with a green fluorescent protein (GFP) tag. A pot experiment was performed to assess the in vivo disease-control efficiency of B. pumilus SQR-N43 and its bio-organic fertilizer. Results indicate that B. pumilus SQR-N43 induced hyphal deformation, enlargement of cytoplasmic vacuoles and cytoplasmic leakage in R. solani Q1 mycelia. A biofilm on the root surface was formed when the roots were inoculated with 10(7)-10(8)cells g(-1) of soil of GFP-tagged B. pumilus SQR-N43. In the pot experiment, the biocontrol reduced the concentration of R. solani. In contrast to applications of only B. pumilus SQR-N43 (N treatment), which produced control efficiencies of 23%, control efficiencies of 68% were obtained with applications of a fermented organic fertilizer inoculated with B. pumilus SQR-N43 (BIO treatment). After twenty days of incubation, significant differences in the number of CFUs and the percentage of spores of B. pumilus SQR-N43 were recorded between the N treatment (2.20×10(7)CFU g(-1) of soil and 79%, respectively) and the BIO treatment (1.67×10(8)CFU g(-1) of soil and 52%, respectively). The results indicate that B. pumilus SQR-N43 is a potent antagonist against R. solani Q1. The BIO treatment was more effective than the N treatment because it stabilized the population and increased the active form of the antagonist.     

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.     

Biocontrol of Rhizoctonia solani AG-2, the causal agent of damping-off by Muscodor cinnamomi CMU-Cib 461

Rhizoctonia solani is a damping-off pathogen that causes significant crop loss worldwide. In this study, the potential of Muscodor cinnamomi, a new species of endophytic fungus for controlling R. solani AG-2 damping-off disease of plant seedlings by biological fumigation was investigated. In vitro tests showed that M. cinnamomi volatile compounds inhibited mycelial growth of pathogens. Among nine solid media tested, rye grain was the best grain for inoculum production. An in vivo experiment of four seedlings, bird pepper, bush bean, garden pea and tomato were conducted. The results indicated that treatment with 30?g of M. cinnamomi inoculum was the minimum dose that caused complete control of damping-off symptoms of all seedlings after one month of planting. The R. solani-infested soil showed the lowest percentage of seed germination. In addition, M. cinnamomi did not cause any disease symptoms. From the results it is clear that M. cinnamomi is effective in controlling R. solani AG-2 both in vitro and in vivo.     

Potential for the integration of biological and chemical control of sheath blight disease caused by Rhizoctonia solani on rice

Biological control using antagonistic microbes to minimize the use of chemical pesticides has recently become more prevalent. In an attempt to find an integrated control system for sheath blight, caused by Rhizoctonia solani in rice, Streptomyces philanthi RM-1-138, commercial formulations of Bacillus subtilis as Larminar^® and B. subtilis strain NSRS 89-24+MK-007 as Biobest^® and chemical fungicides including carbendazim^®, validamycin^®, propiconazole^® and mancozeb^® were applied alone and in combination with S. philanthi RM-1-138. In vitro experiments showed that all treatments tested did provide some control against mycelial growth and sclerotia production by R. solani PTRRS-9. In addition, the four chemical fungicides had no detrimental effects on S. philanthi RM-1-138 even at high concentrations (up to 100 μg/ml). The efficacy of S. philanthi RM-1-138, the commercial formulations of B. subtilis, chemical fungicides alone or in combination with S. philanthi RM-1-138 was also tested in a greenhouse experiment against sheath blight disease on rice plants. All treatments showed some protection of rice for sheath blight by 47–60 % when carbendazim^® was applied alone and up to 74 % when combined with S. philanthi RM-1-138.     

Trichoderma harzianum strain SQR-T37 and its bio-organic fertilizer could control Rhizoctonia solani damping-off disease in cucumber seedlings mainly by the mycoparasitism

Damping-off disease is caused by Rhizoctonia solani and leads to serious loss in many crops. Biological control is an efficient and environmentally friendly way to prevent damping-off disease. Optical micrographs, scanning electron micrographs, and the determination of hydrolytic enzymes were used to investigate the antagonism of Trichoderma harzianum SQR-T37 (SQR-T37) against R. solani. Experiments were performed in pots to assess the in vivo disease-control efficiency of SQR-T37 and bio-organic fertilizer. The results indicate that the mycoparasitism was the main mechanism accounting for the antagonistic activity of SQR-T37. In one experiment, the population of R. solani was decreased from 10⁶ internal transcribed spacer (ITS) copies per gram soil to 10⁴ ITS copies per gram soil by the presence of the antagonist. In this experiment, 45% of the control efficiency was obtained when 8 g of SQR-T37 hyphae per gram soil was applied. In a second experiment, as much as 81.82% of the control efficiency was obtained when bio-organic fertilizer (SQR-T37 fermented organic fertilizer, BIO) was applied compared to only 27.27% of the control efficiency when only 4 g of SQR-T37 hyphae per gram soil was applied. Twenty days after incubation, the population of T. harzianum was 4.12 × 10⁷ ITS copies per gram soil in the BIO treatment, which was much higher than that in the previous treatment (8.77 × 10⁵ ITS copies per gram soil), where only SQR-T37 was applied. The results indicated that SQR-T37 was a potent antagonist against R. solani in a mycoparasitic way that decreased the population of the pathogen. Applying BIO was more efficient than SQR-T37 application alone because it stabilized the population of the antagonist.     

Biocontrol of Rhizoctonia solani damping-off of sugar beet with native Streptomyces strains under field conditions

A 3-year study was conducted to evaluate the effectiveness of two disease-suppressive Streptomyces spp. to control sugar beet Rhizoctonia solani damping off under field conditions. Streptomyces seed treatments reduced seedling damping off in naturally (2005) and artificially (2006 and 2007) infested soils. All biocontrol agents provided better efficacy than Vitavax to control seedling damping-off. There were no significant differences among Streptomyces isolates. Isolate C increased plant stand by 19.5, 50.5 and 53.75% in 2005, 2006 and 2007, respectively. Evaluation of final harvest revealed that the root yield of the biocontrol agents increased compared to untreated control in these years.     

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.     

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.     

Characterization of Genes Involved in Biosynthesis of a Novel Antibiotic from Burkholderia cepacia BC11 and Their Role in Biological Control of Rhizoctonia solani

Genetic manipulation of fluorescent pseudomonads has provided major insight into their production of antifungal molecules and their role in biological control of plant disease. Burkholderia cepacia also produces antifungal activities, but its biological control activity is much less well characterized, in part due to difficulties in applying genetic tools. Here we report genetic and biochemical characterization of a soil isolate of B. cepacia relating to its production of an unusual antibiotic that is very active against a variety of soil fungi. Purification and preliminary structural analyses suggest that this antibiotic (called AFC-BC11) is a novel lipopeptide associated largely with the cell membrane. Analysis of conditions for optimal production of AFC-BC11 indicated stringent environmental regulation of its synthesis. Furthermore, we show that production of AFC-BC11 is largely responsible for the ability of B. cepacia BC11 to effectively control the damping-off of cotton caused by the fungal pathogen Rhizoctonia solani in a gnotobiotic system. Using Tn5 mutagenesis, we identified, cloned, and characterized a region of the genome of strain BC11 that is required for production of this antifungal metabolite. DNA sequence analysis suggested that this region encodes proteins directly involved in the production of a nonribosomally synthesized lipopeptide.