Biocontrol potential of two novel grapevine associated Bacillus strains for management of anthracnose disease caused by Colletotrichum gloeosporioides

A study was conducted to identify bacterial antagonists for biological control of anthracnose which is one of the economically important diseases of grapes. In India, it is caused by Colletotrichum gloeosporioides. Two hundred and ninety-three bacteria were isolated from the grape ecosystem of 43 spatially distant vineyards in peninsular India. Of these, 25 bacteria substantially inhibited the radial growth of C. gloeosporioides in in vitro studies and 18 bacteria significantly reduced infections in vivo. Of these 18 bacteria, 5 and 3 bacteria also significantly reduced percent disease index (PDI) of downy and powdery mildew diseases, respectively. These bacteria were labelled as strains, DR-38, DR-39, TL-171, DRo-198, TS-204, TS-205, and DR-219, and were identified as Bacillus species based on morphological and molecular characterisation. Aqueous suspensions of all these strains applied as foliar sprays at 1×10⁸ cfu/ml on field grown vines significantly lowered the PDI and the AUDPC (area under disease progress curve) of anthracnose when compared with the untreated control, except DRo-198. Strains TS-204 and TL-171 recorded lower PDIs and AUDPC when compared with the other five strains, and TS-204 could effectively suppress ripe rot of berries, too, in vivo. Strains TS-204 and TL-171 are identified for biocontrol of anthracnose in grapes.     

First report of anthracnose caused by Colletotrichum gloeosporioides on Boehravia diffusa in India

Leaf spot symptoms were observed on the leaves of Boehravia diffusa L. plants from different regions of Bilaspur, Himachal Pradesh, India. The symptoms initially appeared as a small light-pale coloured, circular spots enlarge gradually and ultimately leading to drying of the leaves. The disease was found to be caused by a fungus. The fungus was cultured on potato dextrose agar medium and identified as Colletotrichum gloeosporioides. According to the literature, this is thefirst report of anthracnose disease of B. diffusa caused by C. gloeosporioides in India.     

Production of an antifungal protein for control of Colletotrichum lagenarium by Bacillus amyloliquefaciens MET0908

A plant pathogenic fungus, Colletotrichum lagenarium, causing watermelon anthracnose, was isolated from naturally infected leaves, stems, and fruits of watermelon. A bacterial strain, MET0908, showing a potent antifungal activity against C. lagenarium, was isolated from soil. An antifungal protein was purified by 30% ammonium sulfate saturation and concentrated using Centricon 10, DEAE-Sepharose(TM) Fast Flow column and Sephacryl S-100 gel filtration chromatography. The molecular weight of the purified protein was estimated as 40 kDa by SDS-PAGE. The purified protein was stable at 80 degrees C for 20 min and exhibited a broad spectrum of antifungal activity against various plant pathogenic fungi. Confocal microscopy image analysis and scanning electron microscopy showed that the protein acted on the cell wall of C. lagenarium. The purified antifungal protein exhibited beta-1,3-glucanase activity. The N-terminal amino acid sequence of the purified protein was determined as Ser-Lys-Ile-x-Ile-Asn-Ile-Asn-Ile-x-Gln-Ala-Pro-Ala-Pro-x-Ala. A search of the sequence with NCBI BLAST showed no significant homology with any known proteins, suggesting that the purified protein may be novel.     

Integrated control of Colletotrichum gloeosporioides on mango fruit in Taiwan by the combination of Bacillus subtilis and fruit bagging

Anthracnose disease caused by Colletotrichum gloeosporioides in Jingkwang mango grown in Taiwan was significantly reduced by the integration of fruit bagging with either B. subtilis strain LB5 or fungicides. The combined treatments were most effective in reducing early infection during the 2004 season, leading to 56.4 and 58.3% reduction, respectively, while in 2003 reduction accounted for 51 and 52.3%, respectively. Post-harvest application of B. subtilis strain LB5 cell suspensions on fruits already treated by bagging, bagging+LB5 and baggingfungicides in the field reduced anthracnose incidence significantly at all tested concentrations. These results indicate that biocontrol efficacy of B. subtilis LB5 may be due to the prevention of early fruit infection, thereby reducing significantly anthracnose incidence in ripening fruits to much lower levels than those obtained by using a conventional single post-harvest treatment.     

Biological control of anthracnose (Colletotrichum gloeosporioides) in yam by Streptomyces sp.MJM5763

Aim: To find a suitable biocontrol agent for yam anthracnose caused by Colletotrichum gloeosporioides. Methods and Results: An actinobacterial strain, MJM5763, showing strong antifungal activity, multiple biocontrol and plant growth‐promoting traits was isolated from a yam cultivation field in Yeoju, South Korea. Based on morphological and physiological characteristics and analysis of the 16S rDNA sequence, strain MJM5763 was identified as a novel strain of Streptomyces and was designated as Streptomyces sp. MJM5763. Treatment with MJM5763 and the crude culture filtrate extract (CCFE) was effective in suppressing anthracnose in detached yam leaves in vitro and reduced incidence and severity of anthracnose in yam plants under greenhouse conditions. The CCFE treatment was the most effective of all the treatments and reduced the anthracnose severity by 85–88% and the incidence by 79–81%, 90 days after inoculation with the pathogen. CCFE treatment was also effective under field conditions and showed a reduction of 86 and 75% of anthracnose severity and incidence, respectively. Conclusion: Streptomyces sp. strain MJM5763 was effective in biocontrolling anthracnose in yam caused by C. gloeosporioides. Significance and Impact of the Study: Streptomyces sp. MJM5763 is a potential alternative to chemical fungicides for reducing yield losses to anthracnose in yam.     

Adjuvants enhance the biological control potential of an isolate of Colletotrichum gloeosporioides for biological control of sicklepod (Senna obtusifolia)

In greenhouse experiments, unrefined corn oil, Silwet L-77, and an invert emulsion were tested as adjuvants with the mycoherbicidal fungus Colletotrichum gloeosporioides, a weakly virulent pathogen of sicklepod (Senna obtusifolia). A 1:1 (v/v) fungus/corn oil tank mixture containing 0.2% (v/v) Silwet L-77 surfactant reduced the dew period requirements for maximum weed infection and mortality from 16 to 8 h, and delayed the need for free moisture for greater than 48 h. This formulation also resulted in the ability of the pathogen to infect and kill weeds in larger (>5 leaf) growth stages. The invert emulsion resulted in similar effects upon these parameters. These results suggest that invert emulsions, unrefined corn oil and Silwet L-77 surfactant greatly improve the bioherbicidal potential of this pathogen for control of sicklepod, a serious weed pest in the southeastern US.     

Evaluation of Bacillus amyloliquefaciens PEBA20 as a biocontrol agent for Rhizoctonia cerealis and Bipolaris maydis

Rhizoctonia cerealis and Bipolaris maydis are fungal plant pathogens that cause enormous agricultural losses of wheat and maize.Bacillus amyloliquefaciens PEBA20 inhibited mycelial growth and spore germination of the pathogens and reduced fungal infections in wheat and maize, indicating its potential for application as a biocontrol agent.     

Isolation and characterization of Bacillus subtilis KS1 for the biocontrol of grapevine fungal diseases

Bacillus subtilis KS1 was isolated from grape berry skin as a biological control agent against grapevine fungal diseases. KS1 was identified as a new strain of B. subtilis according to morphological, biochemical, and genetic analyses. In vitro bioassay demonstrated that KS1 suppressed the growth of Botrytis cinerea (the casual agent of grape grey mold) and Colletotrichum gloeosporioides (the casual agent of grape ripe rot). The biocontrol activity of KS1 against grapevine fungal diseases in vineyards was evaluated over a 3-year span (from 2007 to 2009). Downy mildew, caused by Plasmopara viticola, was reduced on berry skins and leaves by treatment with KS1. The KS1 genome possesses ituD and lpa-14 genes, both of which play a role in iturin A production followed by iturin A production in the culture. In contrast, mutants lacking both genes lost the antagonistic activity against B. cinerea and C. gloeosporioides and the activity in iturin A production, suggesting that the antagonistic activity of KS1 against grapevine fungal pathogens may depend on iturin A production. As KS1 showed tolerance to various chemical pesticides, chemical pesticides could be applied before and/or after KS1 treatment in vineyards. Due to its potential as a biological control agent against grape downy mildew, KS1 is expected to contribute to the further improvement of integrated pest management systems and to potentially reduce the amount of chemical fungicides applied in vineyards.     

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。     

Purification and characterization of a lipopeptide produced by Bacillus thuringiensis CMB26

AIMS: To isolate an antagonist for use in the biological control of phytopathogenic fungi including Colletotrichum gloeosporioides, then to purify and characterize the biocontrol agent produced by the antagonist. METHODS AND RESULTS: Bacteria that exhibited antifungal activity against the causative agent pepper anthracnose were isolated from soil, with Bacillus thuringiensis CMB26 showing the strongest activity. A lipopeptide produced by B. thuringiensis CMB26 was precipitated by adjusting the pH 2 with 3 n HCl and extracted using chloroform/methanol (2:1, v/v) and reversed-phase HPLC. The molecular weight was estimated as 1447 Da by MALDI-TOF mass spectrometry. Scanning electron and optical microscopies showed that the lipopeptide has activity against Escherichia coli O157:ac88, larvae of the cabbage white butterfly (Pieris rapae crucivora) and phytopathogenic fungi. The lipopeptide had cyclic structure and the amino acid composition was L-Glu, D-Orn, L-Tyr, D-allo-Thr, D-Ala, D-Val, L-Pro, and L-Ile in a molar ratio of 3:1:2:1:1:2:1:1. The purified lipopeptide showed the same amino acid composition as fengycin, but differed slightly in fatty acid composition, in which the double bond was at carbons 13-14 (m/z 303, 316) and there was no methyl group. CONCLUSION: A lipopeptide was purified and characterized from B. thuringiensis CMB26 and found to be similar to the lipopeptide fengycin. This lipopeptide can function as a biocontrol agent, and exhibits fungicidal, bactericidal, and insecticidal activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Compared with surfactin and iturin, the lipopeptide from B. thuringiensis CMB26 showed stronger antifungal activity against phytopathogenic fungi. This lipopeptide is a candidate for the biocontrol of pathogens in agriculture.     

Pathogenicity of Colletotrichum gloeosporioides (Penz.) Sacc. causal agent of anthracnose in different varieties of eggplant (Solanum melongena L.) determined by levels of cross-reactive antigens shared by host and pathogen

Pathogenicity test of the fungal pathogen Colletotrichum gloeosporioides (Penz.) Sacc., causal agent for anthracnose in eggplant (Solanum melongena L.), was performed in 28 commercially cultivated eggplant varieties by analysing the antigenic patterns of host and pathogen. Through initial screening following detached leaf inoculation technique and whole plant inoculation technique, Pusa purple long (Ppl) variety was found to be the most susceptible while Shamala variety (Shav) was the most resistant. Cross-reactive antigens (CRA) shared by susceptible varieties and C. gloeosporioides was detected by immunodiffusion and immunoelectrophoresis and indirect enzyme-linked immunosorbent assay (ELISA). Such antigens could not be detected between the resistant varieties and the pathogen and also between a nonpathogen (Alternaria porri) and all the test varieties. However, ELISA showed that low levels of common antigens were present between all combinations. The level of CRA was found to decrease with increasing resistance. Indirect immunogold labeling followed by silver enhancement revealed that CRA were concentrated mainly in the cell wall regions throughout the tissue. The level of CRA was found to correlate to the pathogenicity of C. gloeosporioides in different eggplant varieties. ELISA may therefore be used to screen the commercially cultivated eggplant varieties for resistance to C. gloeosporioides.     

In vitro,greenhouse and field assessments of cassava lines for resistance to anthracnose disease caused by Colletotrichum gloeosporioides f.sp. manihotis

Fifty-three cassava lines were selected from breeding populations at the International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria and screened in vitro for resistance to cassava anthracnose disease (CAD). The in vitro inoculation of stem cuttings with the fungus Colletotrichum gloeosporioides f.sp. manihotis showed significant differences (p ± 0.05) in acervuli production and in the sensitivity of the cassava lines to the fungal infection after 7 days of incubation at 25 °C. Cassava lines 88/01084, 91/00595, 91/00475, 91/00344, 91/00684, 91/00313, 91/00422, and 91/00344 were highly resistant, with necrotic lesion sizes less than 7 mm. In contrast pedigree lines 88/02549, 89/0008, 91/00390 and 91/00402 were highly susceptible with the largest necrotic lesion size being greater than 20 mm. Ten cassava lines from the in vitro screening that showed varying levels of resistance to CAD were selected, based on their flowering abilities for diallel hydridization trials, and were further screened in greenhouse and field trials for CAD resistance. The greenhouse and field screening showed significant varietal differences (p ± 0.05) in sensitivity to the fungus. In all cases, the progeny lines showed correlated levels of resistance irrespective of the type of screening or assessments. Correlation analysis of the in vitro, greenhouse and field assessments showed that there was a good correspondence among all three methods of evaluating for CAD. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isolation and evaluation of a Bacillus methylotrophicus strain for control of corn stalk rot

Bacteria were isolated from corn plants and efficacy of a selected isolate for control of corn stalk rot caused by Fusarium graminearum was evaluated. The bacterial isolate provided the greatest suppression of F. graminearum in lab studies and reduced corn stalk rot significantly in 2013 and 2014 field experiments.     

Occurrence and impact of the parasitic mite Linobia coccinellae on its host beetle Chrysomela populi: implications for its potential as a biological control agent

Due to social demands with regard to sustainability and ecological considerations, an aim of current research in short-rotation coppices (SRC) is to find effective candidates for biological control of pest species. The great red poplar leaf beetle, Chrysomela populi L. (Coleoptera: Chrysomelidae), is one of the most harmful pests in the SRC. The endoparasitic mite Linobia coccinellae (Scopoli) (Acari: Astigmata, Hemisarcoptidae) lives under the elytra of C. populi and feeds on the beetles' hemolymph. The population dynamics of the mites and their impact on the fecundity and longevity of female beetles were examined in two experiments. To study the occurrence of the mites, beetles were collected from two poplar SRC sites in Germany on three dates. Infestation rates of collected male and female C. populi individuals increased over the course of the year and reached 78–97% in the last survey. Females were parasitized more often than males at any time. For the second part of the study, beetles were reared under laboratory conditions to study the development of the mites and their impact on the hosts. For this purpose, half of the females were actively seeded with five adult females of L. coccinellae and the other half was used as a control. There was no significant impact on longevity or reproductive traits of the beetles. Despite the high rates of infestation, it appears that the mites do not have a serious effect on female beetles. Thus L. coccinellae, by itself, is not an effective biological control agent against C. populi. However, it remains to be investigated whether the mites increase the beetles' susceptibility to other biotic and abiotic factors, and so may contribute to an integrated pest management regime with multiple antagonists.