Isolation of Microbial Antagonists for Biocontrol of Grey Mould Disease of Strawberries

A screening programme is described for the assessment of the potential of biocontrol agents to control grey mould of strawberries caused by Botrytis cinerea. Bacteria were isolated from strawberry fruits, leaves and flowers from a commercial field site and screened for antagonism towards B. cinerea using two in vitro and one in vivo screening techniques. From 559 microorganisms isolated, 108 inhibited pathogen growth on agar plates and 27 of these prevented spore germination on Cellophane membranes. The ability of these 27 isolates to inhibit infection of young strawberry leaves by B. cinerea on whole plants under glass was then tested. Seven isolates reduced grey mould development and were subsequently assessed in a field trial. Two isolates, one of Bacillus pumilus and one of Pseudomonas fluorescens, were as effective or more effective than standard dichlofluanid sprays and may therefore be of potential value as antagonists of B. cinerea.     

抗灰葡萄孢霉菌乳酸菌的筛选

从80株乳酸菌中筛选出45株具有抗灰葡萄孢霉菌活性的乳酸菌菌株,其中10株具有较强抗灰葡萄孢霉菌活性。对这10株乳酸菌菌株的抗植物致病真菌谱进行了研究,这10株乳酸菌对番茄早疫病菌,甜瓜疫霉菌,甜瓜枯萎病菌,苹果炭疽病菌的生长均有较强的抑制作用。其中1株具有广谱抗植物致病真菌活性的乳酸菌菌株BX6-4为植物乳杆菌。经番茄离体叶片接种试验发现,植物乳杆菌BX6-4的发酵液能够在体外强烈地抑制灰葡萄孢霉菌的生长。     

Antagonistic Properties of Two Bacterial Biocontrol Agents of Grey Mould Disease

Bacillus pumilus NCIMB 13374 and Pseudomonas fluorescens NCIMB 13373 inhibit the growth of Botrytis cinerea , the cause of grey mould of strawberries. Both antagonists increased the pH of the growth medium from pH 6 to pH 8-8.5 and both produced antifungal agents. The compound(s) produced by B. pumilus had a fungicidal effect during conidial germination, whereas the compound(s) produced by P. fluorescens had a fungistatic effect. There was no evidence for the production of inhibitory volatile compounds. Both isolates also showed the ability to inhibit other strawberry fungal pathogens, and have potential for a wider range of biocontrol of plant diseases.     

Biological control of grey mould in strawberry fruits by halophilic bacteria

Aims:  Grey mould caused by Botrytis cinerea is an economically important disease of strawberries in Tunisia and worldwide. The aim of this study was to select effective halophilic bacteria from hypersaline ecosystems and evaluate the abilities of antifungal bacteria to secrete extracellular hydrolytic enzymes, anti- Botrytis metabolites and volatiles.
Methods and Results:  Grey mould was reduced in strawberry fruits treated with halophilic antagonists and artificially inoculated with B. cinerea . Thirty strains (20·2%) were active against the pathogen and reduced the percentage of fruits infected after 3 days of storage at 20°C, from 50% to 91·66%. The antagonists were characterized by phenotypic tests and 16S rDNA sequencing. They were identified as belonging to one of the species: Virgibacillus marismortui , B. subtilis , B. pumilus , B. licheniformis , Terribacillus halophilus , Halomonas elongata , Planococcus rifietoensis , Staphylococcus equorum and Staphylococcus sp. The effective isolates were tested for antifungal secondary metabolites.
Conclusions:  Moderately halophilic bacteria may be useful in biological control against this pathogen during postharvest storage of strawberries.
Significance and Impact of the study:  The use of such bacteria may constitute an important alternative to synthetic fungicides. These moderate halophiles can be exploited in commercial production and application of the effective strains under storage and greenhouse conditions.     

Effects of indole-3-acetic acid on Botrytis cinerea isolates obtained from potted plants

We study the growth of different isolates of Botrytis cinerea collected from potted plants which were affected by Botrytis blight in southern Spain during recent years. These isolates, which show widely phenotypic differences when grown in vitro, are differentially affected by growth temperature, gibberellic acid applications and paclobutrazol, an efficient plant growth retardant and fungicide at the same time. In this work, we have evaluated the effect of the auxin indole-3-acetic acid (IAA) dose (0, 1, 10, and 100 mg/plate) on the growth of the collection of B. cinerea isolates obtained from the following potted plants: Cyclamen persicum, Hydrangea macrophylla, Lantona camara, and Lonicera japonica. B. cinerea produces indolacetic acid, but so far the precise biosynthetic pathway and some effects on this fungal species are still unclear, although recent studies have revealed an antifungal activity of IAA on several fungi, including B. cinerea isolated from harvested fruits. Mycelial growth curves and growth rates assessed from difference in colony areas during the both linear and deceleration phase, conidiation (measured as time of appearance), conidia length (microm), and sclerotia production (number/plate) were evaluated in the isolates, which were grown at 26 degrees C on Petri dishes containing potato dextrose agar for up to 35 days. Mycelial growth curves fitted a typical kinetic equation of fungi grown on solid media. B. cinerea isolates showed a high degree of variability in their growth kinetics, depending on the isolate and auxin dose. This plant growth substance delayed mycelial growth during the linear phase in an isolate-dependent manner, thus isolates from C. persicum, H. macrophylla and L. camara were more affected by IAA than L. japonica. On the other hand, 100 mg of IAA was the critical dose to significantly reduce the growth rate in all isolates and to promote brown-striped hyphae development, especially in isolate from C. persicum. 10 and 100 mg IAA delayed conidiation in isolates from H. macrophylla but scarcely effects were found in the conidia length. The sclerotia production process was blocked at IAA doses of 100 mg in isolates from L. camara and L. japonica, and was reduced in isolate from H. macrophylla. However, dose of 100 mg IAA had no effect on sclerotia production in isolate from C. persicum. It was concluded that the effect of IAA on B. cinerea growth depends on the isolate, thus isolates from H. macrophylla and L. camara were the most affected by IAA. B. cinerea reduced its development under IAA applications, depending on the isolate and dose. These results confirm those recently published on the inhibitory effect of IAA on Botrytris species growth.     

百合枯萎病拮抗细菌的筛选、鉴定及其抑菌物质研究

【目的】筛选对百合枯萎病具有抑菌活性的拮抗细菌,对其抑菌活性物质进行初步分离纯化分析。【方法】以强致病力的百合尖孢镰刀菌(Fusarium oxysporum)为靶菌,采用系列稀释法和平板对峙法初筛拮抗细菌,并通过产铁载体能力、水解酶活性、土壤定殖力等多种生防特性指标进行复筛,结合形态学特征、生理生化指标和16S rRNA基因序列比对鉴定其分类地位;利用百合尖孢镰刀菌作为靶菌进行活性追踪,结合酸沉淀、快速柱色谱、HPLC等分离纯化手段,对菌发酵液中的抑菌活性物质进行纯化分析。【结果】在64株百合根际细菌和386株海洋细菌中进行初筛,得到9株对百合镰刀菌具有较强拮抗活性的菌株,最后筛选了1株拮抗活性较强且产铁载体能力和水解酶活性、土壤定殖能力较高的菌株11B91,鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),其抑菌活性物质初步推测可能为iturin和fengycin脂肽类化合物。【结论】菌株11B91在百合枯萎病的生物防治中具有潜在的应用价值,证实海洋来源的微生物也具有防治陆地植物病原菌的潜力,为植物病害的防治拓宽了思路。     

Isolation and characterization of antagonistic Bacillus subtilis strains from the avocado rhizoplane displaying biocontrol activity

AIM: This study was undertaken to isolate Bacillus subtilis strains with biological activity against soil-borne phytopathogenic fungi from the avocado rhizoplane. METHODS AND RESULTS: A collection of 905 bacterial isolates obtained from the rhizoplane of healthy avocado trees, contains 277 gram-positive isolates. From these gram-positive isolates, four strains, PCL1605, PCL1608, PCL1610 and PCL1612, identified as B. subtilis, were selected on the basis of their antifungal activity against diverse soil-borne phytopathogenic fungi. Analysis of the antifungal compounds involved in their antagonistic activity showed that these strains produced hydrolytic enzymes such as glucanases or proteases and the antibiotic lipopeptides surfactin, fengycin, and/or iturin A. In biocontrol trials using the pathosystems tomato/Fusarium oxysporum f.sp. radicis-lycopersici and avocado/Rosellinia necatrix, two B. subtilis strains, PCL1608 and PCL1612, both producing iturin A, exhibited the highest biocontrol and colonization capabilities. CONCLUSIONS: Diverse antagonistic B. subtilis strains isolated from healthy avocado rhizoplanes have shown promising biocontrol abilities, which are closely linked with the production of antifungal lipopeptides and good colonization aptitudes. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the few reports dealing with isolation and characterization of B. subtilis strains with biocontrol activity against the common soil-borne phytopathogenic fungi F. oxysporum f.sp. radicis-lycopersici and R. necatrix.     

灰葡萄孢强除草菌株的筛选与活性物质的组分分析

灰葡萄孢产生的毒素具有较强的除草活性,开发成生物除草剂前景广阔。本试验以灰葡萄孢BC4为出发菌株,通过紫外线和化学诱变剂诱变分生孢子或菌丝体,获得了所产毒素除草活性比野生菌株显著增强的3个突变菌株。对各菌株产生的毒素提取液进行HPLC分析,发现获得的3个突变菌株中各个组分的含量均显著高于野生菌株,且遗传稳定。利用HPLC制备得到了4个组分,其中组分Ⅰ、Ⅳ对马唐和反枝苋具有很强的杀除活性,为灰葡萄孢的遗传改良及毒素源除草剂的工业化生产奠定了基础。     

Bioassay methods for the detection of antifungal activity by Pseudomonas antimicrobica against the grey mould pathogen Botrytis cinerea

Antagonism against the grey mould pathogen Botrytis cinerea by Pseudomonas antimicrobica was demonstrated in vitro and in vivo. Cell-free filtrates showed activity against B. cinerea growing on Potato Dextrose Agar (PDA) in a media-dependent manner with the most distinct antagonism being produced in Czapek Dox Broth (CDB). Cell-free filtrates of CDB-grown cultures also significantly reduced conidial germination of B. cinerea. An assays based on the inhibition of conidial germination was compared with two assays measuring the antagonism of mycelial growth on PDA. The conidial germination bioassay was more sensitive in the detection of this antifungal activity than the Petri dish bioassay while a bioassay using Microdetection plates did not detect antagonism due to the small loading capacity of the latter. The conidial germination bioassay was modified for detection of antibiosis on the surface of strawberry leaves. Significant reductions in percentage conidial germination were recorded on the surface of leaves of both micropropagated and glasshouse grown strawberry plants when the antifungal compounds of Ps. antimicrobica were applied to the leaf tissue with the conidia. In addition, antifungal compounds were also detectable when conidia were applied to leaf tissue which had previously been sprayed with cells of Ps. antimicrobica. These tests indicate that Ps. antimicrobica would be a suitable biocontrol agent for the control of B. cinerea.     

Screening of free-living rhizospheric bacteria for their multiple plant growth promoting activities

Plant growth promoting rhizobacteria (PGPR) are known to influence plant growth by various direct or indirect mechanisms. In search of efficient PGPR strains with multiple activities, a total of 72 bacterial isolates belonging to Azotobacter, fluorescent Pseudomonas, Mesorhizobium and Bacillus were isolated from different rhizospheric soil and plant root nodules in the vicinity of Aligarh. These test isolates were biochemically characterized. These isolates were screened in vitro for their plant growth promoting traits like production of indoleacetic acid (IAA), ammonia (NH(3)), hydrogen cyanide (HCN), siderophore, phosphate solubilization and antifungal activity. More than 80% of the isolates of Azotobacter, fluorescent Pseudomonas and Mesorhizobium ciceri produced IAA, whereas only 20% of Bacillus isolates was IAA producer. Solubilization of phosphate was commonly detected in the isolates of Bacillus (80%) followed by Azotobacter (74.47%), Pseudomonas (55.56%) and Mesorhizobium (16.67%). All test isolates could produce ammonia but none of the isolates hydrolyzed chitin. Siderophore production and antifungal activity of these isolates except Mesorhizobium were exhibited by 10-12.77% isolates. HCN production was more common trait of Pseudomonas (88.89%) and Bacillus (50%). On the basis of multiple plant growth promoting activities, eleven bacterial isolates (seven Azotobacter, three Pseudomonas and one Bacillus) were evaluated for their quantitative IAA production, and broad-spectrum (active against three test fungi) antifungal activity. Almost at all concentration of tryptophan (50-500 microg/ml), IAA production was highest in the Pseudomonas followed by Azotobacter and Bacillus isolates. Azotobacter isolates (AZT(3), AZT(13), AZT(23)), Pseudomonas (Ps(5)) and Bacillus (B(1)) showed broad-spectrum antifungal activity on Muller-Hinton medium against Aspergillus, one or more species of Fusarium and Rhizoctonia bataticola. Further evaluation of the isolates exhibiting multiple plant growth promoting (PGP) traits on soil-plant system is needed to uncover their efficacy as effective PGPR.     

Identification of Botrytis cinerea susceptibility loci in Arabidopsis thaliana

Botrytis cinerea is a major pathogen of fruit and vegetable crops causing both pre- and post-harvest grey mould. We have analysed 16 Arabidopsis thaliana ecotypes for natural variation in B. cinerea susceptibility. Susceptibility was associated with lower camalexin accumulation, and three ecotypes (Cape Verdi Islands (Cvi-0), Slavice (Sav-0) and Kindalville (Kin-0)) showed differential susceptibility to the two B. cinerea isolates used. Subsequently, to better understand the genetic control of grey mould disease, we assayed the Arabidopsis Landsberg erecta (Ler) x Columbia (Col-0) recombinant inbred population with the two isolates, and identified multiple small-to-medium-effect quantitative trait loci (QTL) governing susceptibility. Interestingly, the QTL for each isolate are distinct, suggesting that different mechanisms govern defence against these two isolates. Two QTL for each isolate exhibited epistatic interactions with specific allele combinations generating heightened B. cinerea susceptibility.     

Identification and characterization of a novel Bacillus subtilis strain with potent antifungal activity of a flagellin-like protein

The filamentous fungus Botrytis cinerea is an important agricultural pathogen affecting a wide range of cultivated plants. Since World War II, chemical fungicides have been the go-to method for agricultural pathogen control. However, the potential adverse environmental and health effects of these chemicals have led to an increasing demand for alternative methods of pathogen control, including biological control agents. In this study, we identified a bacterial isolate with strong antagonistic activity against B. cinerea. An analysis of the 16S rRNA gene sequence for this isolate identified it as a novel strain of Bacillus subtilis. Culture media from this isolate were harvested and fractionated using ion exchange and gel filtration chromatography. The fraction exhibiting the highest level of antifungal activity was identified, and its sequence determined by electrospray tandem mass spectrometry had significant similarity to flagellin. This flagellin-like protein was exogenously expressed in Escherichia coli, and screened for antifungal activity against B. cinerea. This flagellin-like protein demonstrated clear antifungal activity of inhibiting B. cinerea growth.     

烟草疫霉拮抗菌枯草芽孢杆菌21b菌株的分离鉴定及其生物学特性研究

【目的】为了控制烟草疫霉(Phytophthora nicotianae)引起的烟草黑胫病对烟草生产造成的危害。【方法】采用稀释平板法从贵州省毕节地区烟草根际土壤中分离筛选拮抗烟草疫霉的细菌菌株,然后经形态观察、Biolog鉴定及16S rRNA基因序列分析,对分离菌株进行鉴定,同时测定抗菌谱,单因子变量分析、优化生长条件。【结果】共分离得到44株拮抗烟草疫霉的细菌菌株,其中菌株21b对烟草黑胫病菌菌丝生长的抑制率达78.33%,鉴定为枯草芽孢杆菌(Bacillus subtilis)。该菌株对烟草青枯病菌(Ralstonia solanacearum)、烟草灰霉病菌(Botrytis cinerea)、烟草赤星病菌(Alternaria alternate)和烟草炭疽病菌(Colletotrichum destructivum)均具有拮抗作用,抑菌圈大小分别为19.5、18.2、14.6和13.4 mm,最佳的发酵条件为:温度30°C、p H 7.0–8.0、装液量12%、盐浓度0.5%。【结论】分离筛选到一株对烟草寄生疫霉有较强拮抗活性的细菌菌株,为进一步开发烟草黑胫病的生防菌剂提供了菌种资源。     

牡丹根部内生细菌的分离鉴定及脂肽类物质的拮抗活性研究

【目的】从牡丹(Paeonia suffruticosa Andr.)根部组织中分离鉴定内生细菌,测定拮抗菌株脂肽类活性物质的体外抑菌活性。【方法】采用平板对峙法筛选出对牡丹灰霉病菌(Botrytis paeoniae Oadem)、牡丹炭疽病菌(Gloeosporium sp.)、牡丹黑斑病菌(Altenaria sp.)、牡丹黄斑病菌(Phyllosticta commonsii)有拮抗作用的内生细菌。基于形态特征、生理生化特性和16S rRNA基因序列同源性鉴定拮抗菌株。根据脂肽类抗菌物质合成相关基因序列对拮抗菌株进行基因扩增检测,采用酸沉淀法提取拮抗菌株的脂肽类物质,平板对峙法测定脂肽类物质的体外抑菌活性。【结果】从牡丹根部组织中共分离获得62株内生细菌,其中菌株Md31和Md33对4种病原菌均有较明显的抑制作用。Md31和Md33被鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。通过对菌株Md31和Md33进行5个脂肽类合成功能基因bmyB、fenD、ituC、srfAA和srfAB的检测,序列同源性分析,表明两个菌株具有合成脂肽类物质的能力。菌株Md31和Md33的脂肽类粗提物对所测试的牡丹病原真菌均具有不同程度的抑制作用。【结论】获得了2株对牡丹病原菌有良好抑制效果的解淀粉芽孢杆菌Md31和Md33,两个菌株的脂肽类粗提物也具有较强的体外抑菌活性,该研究为牡丹内生细菌的进一步开发应用奠定了基础。     

Characterization and antimicrobial activity of the bioactive metabolites in streptomycete isolates

Twenty different streptomycete isolates were obtained from soils of southeast Serbia. Five isolates identified as Streptomyces hygroscopicus (SH100, SH101, SH102, SH103, and SH104) showed strong activity against Botrytis cinerea, a parasite found in domestic vines. These isolates were extensively studied for their in vitro antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, yeasts and fungi, and also antiviral activity against Herpes simplex. The results indicated that the obtained isolates were highly active against Botrytis cinerea, Candida albicans, and Herpes simplex, with an inhibition zone of approximately 31 mm. The structure of the bioactive components was determined using elemental analysis, as well as UV/VIS, FTIR, and TLC.     

Antifungal cyclopeptolide from fungal saprophytic antagonist Ulocladium atrum

The saprophytic fungus Ulocladium atrum Preuss is a promising biological control agent for Botrytis cinerea in greenhouse- and field-grown crops. However, despite its known potent antifungal activity, no antifungal substance has yet been reported. In an effort to characterize the antifungal substance from U atrum, we isolated an antibiotic peptide. Based on extensive spectroscopic analyses, its structure was established as a cyclopeptolide with a high portion of N-methylated amino acids, and its 1H and 13C chemical shifts were completely assigned based on extensive 1D and 2D NMR experiments. Compound 1 exhibited potent antifungal activity against the plant pathogenic fungus Botrytis cinerea and moderate activity against Alternaria alternate and Magnaporthe grisea.     

Secondary metabolites influence Arabidopsis/Botrytis interactions: variation in host production and pathogen sensitivity

Numerous studies have suggested that plant/pathogen interactions are partially mediated via plant secondary metabolite production and corresponding pathogen tolerance. However, there are inconsistent reports on the ability of particular compounds to provide resistance to a pathogen. Most of these studies have focused on individual isolates of a given pathogen, suggesting that pathogens vary in their sensitivity to plant-produced toxins. We tested variability in virulence among pathogen isolates, and the impact on this by plant production of, and pathogen tolerance to, secondary metabolites. Botrytis cinerea isolates showed differing sensitivity to purified camalexin, and camalexin-sensitive isolates produced larger lesions on camalexin-deficient Arabidopsis genotypes than on the wild type. In contrast, the camalexin-insensitive isolate produced lesions of similar size on wild-type and camalexin-deficient Arabidopsis. Additional analysis with Arabidopsis secondary metabolite biosynthetic mutants suggests that Botrytis also has variable sensitivity to phenylpropanoids and glucosinolates. Furthermore, Botrytis infection generates a gradient of secondary metabolite responses emanating from the developing lesion, with the Botrytis isolate used determining the accumulation pattern. Collectively, our results indicate that Arabidopsis/Botrytis interactions are influenced at the metabolic level by variations in toxin production in the host and sensitivity in the pathogen.     

Antifungal activity of valinomycin, a peptide antibiotic produced by Streptomyces sp. Strain M10 antagonistic to Botrytis cinerea

A strain of Streptomyces sp. (M10) antagonistic to Botrytis cinerea was isolated from orchard soil obtained from Jeju Island, Korea. An antifungal substance (CN1) was purified from the culture extracts of the strain, and then identified as valinomycin through extensive spectroscopic analyses. Valinomycin showed potent in vitro antifungal activity against Botrytis cinerea and also in vivo control efficacy against Botrytis blight development in cucumber plants. Overall, the disease control efficacy of valinomycin was similar to that of vinclozolin, a commercial fungicide. This study provides the first report on the disease control efficacy of valinomycin against Botrytis blight.     

Antagonistic actinomycetes from Moroccan soil to control the grapevine gray mold

One hundred and forty-two different actinomycete strains were isolated from rhizosphere soil of Vitis vinifera L. sampled from four Moroccan areas. To evaluate the antifungal effect of the different collected actinomycete isolates, five fungi known to be phytopathogens (Pythium ultimum, Fusarium oxyysporum f. sp. albedinis, Sclerotium rolfsii, Verticillium dahliae and Botrytis cinerea) were used. Results showed that 24 isolates had an in vitro inhibitory effect toward at least 4 of the indicator fungi, but only 9 inhibited all these phytopathogens. These nine isolates were subsequently evaluated individually using in vitro grapevine plantlets for their ability to protect against plant gray mold. We demonstrate here that pre-inoculation of plantlets with these isolates allow them to withstand Botrytis cinerea. Six of these strains were shown to belong to the genus Streptomyces and three to the genus Micromonospora. These findings indicate the potential of developing effective actinomycetes from Moroccan habitats for the biological control of Botrytis cinerea. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

番茄灰霉菌拮抗放线菌LA-5的筛选及鉴定

利用稀释涂布法从番茄根际土壤中分离放线菌,并以番茄灰霉菌为靶标,利用对峙培养法和牛津杯法筛选拮抗放线菌,得到一株具有较强抑菌活性的放线菌LA-5.通过培养特征、生理生化特性及基于16S rDNA 序列系统进化分析,将菌株LA-5初步鉴定为链霉菌.复筛结果显示,LA-5发酵滤液对番茄灰霉菌孢子萌发及菌丝生长均有明显的抑制作用,其中100倍发酵滤液对孢子萌发抑制率和菌丝生长抑制率均在50%以上;受抑制菌落呈白色,气生菌丝萎缩稀疏,菌丝纤细、分支明显减少.离体防效试验显示,菌株LA-5发酵原液对番茄灰霉病防效可达83.4%.该菌株有望开发为防治番茄灰霉病的生防菌株.