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.     

Biocontrol of Rhizoctonia solani Damping-Off of Tomato with Bacillus subtilis RB14

Bacillus subtilis RB14, which showed antibiotic activities against several phytopathogens in vitro by producing the antibiotics iturin A and surfactin, was subjected to a pot test to investigate its ability to suppress damping-off of tomato seedlings caused by Rhizoctonia solani. To facilitate recovery from soil, B. subtilis RB14-C, a spontaneous streptomycin-resistant mutant of RB14, was used. Damping-off was suppressed when the culture broth, cell suspension, or cell-free culture broth of RB14-C was inoculated into soil. Iturin A and surfactin were recovered from the soils inoculated with the cell suspension of RB14-C, confirming that RB14-C produced them in soil. The gene lpa-14, which was cloned from RB14 and required for the production of both antibiotics, was mutated in RB14-C, and a mutant, R(Delta)1, was constructed. The level of disease suppressibility of R(Delta)1 was low, but R(Delta)1(pC115), a transformant of R(Delta)1 with the plasmid pC115 carrying lpa-14, was restored in suppressibility. These results show that the antibiotics iturin A and surfactin produced by RB14 play a major role in the suppression of damping-off caused by R. solani. RB14-C, R(Delta)1, and R(Delta)1(pC115) persisted in soil during the experimental period and were recovered from the soil, mostly as spores.     

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.     

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.     

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 and promotion of tomato plant growth by endophytic actinomycetes isolated from native plants of Algerian Sahara

Thirty-four endophytic actinomycetes were isolated from the roots of native plants of the Algerian Sahara. Morphological and chemical studies showed that twenty-nine isolates belonged to the Streptomyces genus and five were non-Streptomyces. All isolates were screened for their in vitro antifungal activity against Rhizoctonia solani. The six that had the greatest pathogen inhibitory capacities were subsequently tested for their in vivo biocontrol potential on R. solani damping-off in sterilized and non-sterilized soils, and for their plant-growth promoting activities on tomato seedlings. In both soils, coating tomato seeds with antagonistic isolates significantly reduced (P < 0.05) the severity of damping-off of tomato seedlings. Among the isolates tested, the strains CA-2 and AA-2 exhibited the same disease incidence reduction as thioperoxydicarbonic diamide, tetramethylthiram (TMTD) and no significant differences (P < 0.05) were observed. Furthermore, they resulted in a significant increase in the seedling fresh weight, the seedling length and the root length of the seed-treated seedlings compared to the control. The taxonomic position based on 16S rDNA sequence analysis and phylogenetic studies indicated that the strains CA-2 and AA-2 were related to Streptomyces mutabilis NBRC 12800^T (100% of similarity) and Streptomyces cyaneofuscatus JCM 4364^T (100% of similarity), respectively.     

Stilbella aciculosa: A Potential Biocontrol Fungus against Rhizoctonia solani

A semi‐solid fermentation product of the potential biocontrol fungus Stilbella aciculosa was formulated on wheat bran: water (1:1, w/w) and incubated 5, 10 and 15 days before addition to soil infested with the pathogen Rhizoctonia solani. Generally, preparations did not reduce survival of the pathogen in infested beet seed but they did prevent saprophytic growth of the pathogen from beet seed into soil. The magnitude of reduction by the 15‐day‐old inoculum was greater than that by the 5‐day‐old inoculum. Ten‐day‐old bran preparations of S. aciculosa at rates of 0.5 and 1.0% (w/w) in soil prevented post‐emergence damping‐off of cotton, radish and sugar beet in the glasshouse and a rate of 1.0% gave stands similar to those in the non‐infested control soil. The antagonist, grown on perlite formulated with molasses, cornmeal, alfalfa tissue or corn stover, prevented damping‐off of cotton in a naturally infested soil. However, the stands were not as great as that in soil planted with pentachloronitrobenzene (PCNB)‐treated seed. Toxic metabolites, produced by S. aciculosa developing on various substrates, slightly inhibited the growth of R. solani in culture and induced cytoplasmic leakage of the pathogen mycelium.     

Induction of defense response against Rhizoctonia solani in cucumber plants by endophytic bacterium Bacillus thuringiensis GS1

An endophytic bacterium, Bacillus thuringiensis GS1, was isolated from bracken (Pteridium aquilinum) and found to have maximal production of chitinase (4.3 units/ml) at 5 days after culture. This study investigated the ability of B. thuringiensis GS1 to induce resistance to Rhizoctonia solani KACC 40111 (RS) in cucumber plants. Chitinase activity was greatest in RS-treated plants at 4 days. beta-1,3- Glucanase activity was highest in GS1-treated plants at 5 days. Guaiacol peroxidase (GPOD) activity increased continuously in all treated plants for 5 days. Ascorbate peroxidase (APX) activity in RS-treated plants was increased 1.5-fold compared with the control at 4 days. Polyphenol oxidase (PPO) activity in RS-treated plants was increased 1.5-fold compared with the control at 3 days. At 5 days after treatment, activity staining revealed three bands with chitinase activity (Ch1, Ch2, and Ch3) on SDSPAGE of cucumber plants treated with GS1+RS, whereas only one band was observed for RS-treated plants (Ch2). One GPOD isozyme (Gp1) was also observed in response to treatment with RS and GS1+RS at 4 days. One APX band (Ap2) was present on the native-PAGE gel of the control, and GS1- and GS1+RS-treated plants at 1 day. PPO bands (Po1 and Po2) from RS- and GS1+RS-treated plants were stronger than in the control and GS1-treated plants upon native-PAGE at 5 days. Taken together, these results indicate that the induction of PR proteins and defense-related enzymes by B. thuringiensis GS1 might have suppressed the damping-off caused by R. solani KACC 40111 in cucumber plants.     

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.     

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.     

Application of endophytic bacteria for the biocontrol of Rhizoctonia solani (Cantharellales: ceratobasidiaceae) damping-off disease in cotton seedlings

The antifungal potentialities of three endophytic bacterial strains, Stenotrophomonas maltophila H8 (Xanthomonadales: Xanthomonadaceae), Pseudomonas aeruginosa H40 (Pseudomonadales: Pseudomonadaceae) and Bacillus subtilis H18 (Bacillales: Bacillaceae) were evaluated against the phytopathogenic fungus Rhizoctonia solani in cotton seedlings under greenhouse conditions. The bacterial strains were applied as a soil drench or talc-based bioformulation in R. solani-infested soil and non-infested soil. Results indicated that the soil drench treatment was more efficient than talc-based bioformulation. A significant increase of seed emergence and seedling survival with a clear reduction of disease severity was achieved with the endophytic bacterial treatments. At the same time, the fresh weight, dry weight, shoot length and root length of the treated plants were markedly enhanced. Moreover, there was an apparent induction of the antioxidant enzymes (peroxidase, polyphenol oxidase and catalase) of the treated seedlings. Gas chromatography–mass spectrometry revealed the presence of various bioactive compounds in the bacterial supernatant. The antagonistic activity of the bacterial strains against R. solani was attributed to their capability to produce a broad spectrum of antifungal compounds in addition to bioactive molecules that can trigger the systemic resistance in the infected seedlings.     

Powder formulation of Burkholderia cepacia for control of rape seed damping-off caused by Rhizoctonia solani

Talc-based formulation of Burkholderia cepaci strain Bu1 was tested as seed and soil drenchs separately for its ability to control Rhizoctonia soloni the causal agent of rape seed damping-off in greenhouse and field trials. In general, the formulated bacteria was more effective to suppress the disease than the suspension of bacteria cells in carboxymethylcellulose solution (1% w/v), in both greenhouse and field trials. The formulation of strain Bul as soil and seed treatments had the greatest effect on reducing the rape seed damping-off in greenhouse and field trials (66.7, 53.3, 64.4 and 40% respectively). The formulation of strain Bu1 as soil and seed treatments were the most effective treatments to increase the root dry weights in the infected soil in greenhouse. The formulation of strain Bul as soil drench had the greatest effect on enhancement of the fresh weight of roots and stem fresh and dry weights. The formulation of strain Bu1 stored at 4 degrees C exhibited better shelf Life and efficacy in vitro than it's counterpart stored at 25 degrees C.     

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.     

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.     

The Effect of Mode of Application of Bacillus subtilis RB14-C on its Efficacy as a Biocontrol Agent Against Rhizoctonia solani

Bacillus subtilis RB14‐C, which produces the antibiotic iturin A, was investigated for its effectiveness as a biocontrol agent against Rhizoctonia solani infecting tomato using seed coating and/or direct introduction of the bacteria to the soil. The ability of RB14‐C to colonize plant roots and produce iturin A in soil, depending on the method of bacterial application, was also determined. Seed coating and the combined treatment (soil and seed bacterization) did not protect seedlings against damping‐off caused by R. solani. By contrast, RB14 introduced only to the soil controlled the disease. The total number of RB14‐C bacteria on the roots of plants grown from coated seeds was significantly lower than on the roots of plants grown in soil mixed with the bacteria. In the combined treatment, application of B. subtilis with seeds to soil preinoculated with this bacterium, at first suppressed the population of RB14‐C in the soil. Then the colonization was generally uniform. The concentration of iturin A in non‐planted soil was highest at the beginning of the experiment (i.e. after application of the bacterial suspension) but then decreased, and was undetectable 3 days after incubation. However, after seed planting the antibiotic was produced again around young roots. Bacteria introduced to the soil as a seed coating also released the antibiotic around the seeds.     

Biocontrol of Rhizoctonia solani and Pythium ultimum on Capsicum by Trichoderma koningii in potting medium.

Two isolates of Trichoderma koningii were evaluated for efficacy in control of damping-off diseases in seedlings of Capsicum annuum grown in pasteurized potting medium in a glasshouse. A selected isolate of binucleate Rhizoctonia and two fungicides were also included as standards for control of Rhizoctonia solani and Pythium ultimum var. sporangiiferum. Both isolates of T. koningii reduced seedling death caused by R. solani in one of two experiments, and by P. u. sporangii-ferum in two of three experiments. Neither isolate of T. koningii suppressed damping-off caused by either pathogen as consistently as the binucleate Rhizoctonia or fungicides. The implications of these results for commercial disease management are discussed.     

一株抗水稻纹枯病菌的解淀粉芽胞杆菌分离与鉴定

【目的】筛选对水稻纹枯病菌(Rhizoctonia solani)具有强拮抗作用的细菌菌株。【方法】用指示菌法筛选拮抗菌株;通过形态观察、生理生化实验、Biolog及16S rDNA序列分析鉴定目标菌株;利用平板双向培养法和滤纸片扩散法测定抑菌谱及拮抗性质。【结果】分离到一株高活力的水稻纹枯病菌拮抗菌株YB-3,该菌株属于解淀粉芽胞杆菌(Bacillus amyloliquefaciens);菌株YB-3对常见的14株病原真菌和7株细菌具有较强的拮抗作用,并发现其对亲缘关系较近的芽孢菌属有较强的拮抗作用;该菌株的抑制活性具有温度稳定、耐酸、但对蛋白酶敏感的特点。【结论】通过指示菌法筛选到一株对水稻纹枯病菌有强拮抗作用的解淀粉芽胞杆菌(B.amyloliquefaciens)YB-3,它具有广谱、高效的植物病原菌拮抗活性。     

Scanning electron microscopic examination of a pellet formulation of Bacillus megaterium and B. pumilus,antagonists of Rhizoctonia solani,and survival during storage

Bacterial formulations, produced using both Bacillus megaterium and B. pumilus individually with pharmaceutical technology, were formulated using a wet granular method. Viability testing in the laboratory revealed that bacterial populations rapidly declined during storage at room temperature (26–30 °C) for 6 months. The scanning electron microscope (SEM) was used to observe bacterial formulations. Both endospores and vegetative cells of B. megaterium and B. pumilus were detected on the formulation surfaces. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

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.