Antifungal activity of rhizospheric bacteria

Fluorescent Pseudomonad spp. were isolated from the rhizosphere of potato plants (Algeria) by serial dilutions of rhizosphere soils on Kings B medium and were tested for their antifungal activity. The antifungal activity of the Pseudomonas isolated from Potatoes rhizosphere was tested against Pythium ultimum, Rhizoctonia solani and Fusarium oxysporum in dual culture with bacteria on PDA. The Petri dish was divided into tow, on one the bacteria was spread and on the opposite side fungal plugs were inoculated and incubated for one week. Fourteen bacteria were isolated; only one isolate inhibited the growth of Pythium ultimum, Rhizoctonia solani, Fusarium solani; Fusarium oxysporum f.sp. albedinis and Fusarium oxysporum f. sp. Lycopersici with inhibition zones of 39.9, 33.7, 30.8, 19.9 and 22.5 mm respectively.     

Production of macrosphelide A by the mycoparasite Coniothyrium minitans

Coniothyrium minitans, a mycoparasite of sclerotia of Sclerotinia sclerotiorum and Sclerotium cepivorum, produced four closely related metabolites inhibitory to fungal growth. The major metabolite, identified as macrosphelide A, had IG(50) values (the concentration of metabolite to inhibit growth by 50%) of 46.6 and 2.9 microgram ml(-1) against S. sclerotiorum and S. cepivorum, respectively. This is the first report of both antifungal activity due to macrosphelide A as well as isolation of macrosphelide A from C. minitans.     

大豆内生细菌的分离及根腐病拮抗菌的筛选鉴定

内生细菌存在于健康植物体内,一些内生细菌具有促生长、抗病和固氮等生物学功能.本项研究采用化学药剂表面灭菌方法从黑龙江省大豆品种合丰25的根、茎、叶和种子中分离到大量内生细菌,其种群数量在根部最多,为3.4×103CFU/g,在叶部次之,为2.8×103CFU/g,在茎部和种子中最少,为2.9×102 CFU/g和1.4×102CFU/g.从121株内生细菌中筛选到31株对大豆根腐病菌Fusarium oxysporum f. sp.soybean具有较强抑制作用的拮抗内生细菌,其中菌株TF28抑菌谱广,抑菌率高,对不同植物的病原菌F. oxysporum的抑菌率为80.2%～96.7%.经形态、生理生化和16S rRNA鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens).     

Evaluation of Nostoc strain ATCC 53789 as a potential source of natural pesticides

The cyanobacterium Nostoc strain ATCC 53789, a known cryptophycin producer, was tested for its potential as a source of natural pesticides. The antibacterial, antifungal, insecticidal, nematocidal, and cytotoxic activities of methanolic extracts of the cyanobacterium were evaluated. Among the target organisms, nine fungi (Armillaria sp., Fusarium oxysporum f. sp. melonis, Penicillium expansum, Phytophthora cambivora, P. cinnamomi, Rhizoctonia solani, Rosellinia, sp., Sclerotinia sclerotiorum, and Verticillium albo-atrum) were growth inhibited and one insect (Helicoverpa armigera) was killed by the extract, as well as the two model organisms for nematocidal (Caenorhabditis elegans) and cytotoxic (Artemia salina) activity. No antibacterial activity was detected. The antifungal activity against S. sclerotiorum was further studied with both extracts and biomass of the cyanobacterium in a system involving tomato as a host plant. Finally, the herbicidal activity of Nostoc strain ATCC 53789 was evaluated against a grass mixture. To fully exploit the potential of this cyanobacterium in agriculture as a source of pesticides, suitable application methods to overcome its toxicity toward plants and nontarget organisms must be developed.     

Bacillus strains isolated from rhizosphere showed plant growth promoting and antagonistic activity against phytopathogens

Seven bacterial isolates screened from rhizosphere of common bean growing at Uttarakhand Himalaya showed potential plant growth promoting (PGP) and antagonistic activities. Based on 16S rRNA gene sequence the isolate BPR7 was identified as Bacillus sp. BPR7. The strain BPR7 produced IAA, siderophore, phytase, organic acid, ACC deaminase, cyanogens, lytic enzymes, oxalate oxidase, and solubilized various sources of organic and inorganic phosphates as well as potassium and zinc. Strain BPR7 strongly inhibited the growth of several phytopathogens such as Macrophomina phaseolina, Fusarium oxysporum, F. solani, Sclerotinia sclerotiorum, Rhizoctonia solani and Colletotricum sp. in vitro. Cell-free culture filtrate of strain BPR7 also caused colony growth inhibition of all test pathogens. PGP and antifungal activities of Bacillus sp. BPR7 suggest that it may be exploited as a potential bioinoculant agent for P. vulgaris.     

Effects on growth and comparison of root tissue colonization patterns of Eucalyptus viminalis by pathogenic and nonpathogenic strains of Fusarium oxysporum

Soilborne pathogens, especially Fusarium oxysporum , are responsible for damping-off and root necrosis in Eucalyptus nurseries. New technologies are increasingly considering strategies for plant disease control other than chemical fungicides. Among these, natural fungal antagonists, which are colonizers of the root cortex, are potential biocontrol agents. An in vitro system was used: (1) to test the pathogenic effects of F. oxysporum strain Foeu1 which was recovered from a forest nursery soil; (2) to explore the potential of the nonpathogenic F. oxysporum strain Fo47, which is known for its efficiency in biological control, to suppress damping-off of Eucalyptus seedlings; (3) to compare the patterns of root colonization and host response to invasion by the two Fusarium strains inoculated separately in a time-course study. Root inoculation of E. viminalis with F. oxysporum strain Foeu1 caused damping-off in young seedlings in vitro , whilst disease symptoms were not visible in plants inoculated with F. oxysporum strain Fo47 or when both strains (Foeu1 + Fo47) were inoculated simultaneously. Each strain showed similarities in patterns of root tissue colonization, and in the processes of root penetration and initial colonization. Differential effects on root tissue were observed with fungal development within the cortex: ingress of strain Foeu1 was accompanied by severe host-cell alterations whilst no tissue damage occurred with development of strain Fo47.     

Antifungal activity of Bacillus amyloliquefaciens NJN-6 volatile compounds against Fusarium oxysporum f. sp. cubense

Bacillus amyloliquefaciens NJN-6 produces volatile compounds (VOCs) that inhibit the growth and spore germination of Fusarium oxysporum f. sp. cubense. Among the total of 36 volatile compounds detected, 11 compounds completely inhibited fungal growth. The antifungal activity of these compounds suggested that VOCs can play important roles over short and long distances in the suppression of Fusarium oxysporum.     

Pn-AMPs,the hevein-like proteins from Pharbitis nil confers disease resistance against phytopathogenic fungi in tomato,Lycopersicum esculentum

The antifungal activity of hevein-like proteins has been associated with their chitin-binding activities. Pn-AMP1 and Pn-AMP2, two hevein homologues from Pharbitis nil, show in vitro antifungal activities against both chitin and non-chitin containing fungi. Purified Pn-AMPs retained antifungal activities only under non-reducing conditions. When Pn-AMP2 cDNA was constitutively expressed in tomato (Lycopersicon esculentum) plants under the control of CaMV35S promoter, the transgenic plants showed enhanced resistance against both the non-chitinous fungus Phytophthora capsici, and the chitin-containing fungus Fusarium oxysporum. Thus, the chitin component in the fungal cell wall is not an absolute requirement for Pn-AMP's antifungal activities. These results when considered together suggest that Pn-AMPs have the potential for developing transgenic plants resistant to a wide range of phytopathogenic fungi.     

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.     

Antifungal activity and enhancement of plant growth by Bacillus cereus grown on shellfish chitin wastes

Bacillus cereus QQ308 produced antifungal hydrolytic enzymes, comprising chitinase, chitosanase and protease, when grown in a medium containing shrimp and crab shell powder (SCSP) produced from marine waste. The growth of the plant-pathogenic fungi Fusarium oxysporum, Fusarium solani, and Pythium ultimum were considerably affected by the presence of the QQ308 culture supernatant. The supernatant inhibited spore germination and germ tube elongation of F. oxysporum, F. solani, and P. ultimum. The increase in the growth time of the fungal culture was associated with a gradual decrease in inhibition. Besides antifungal activity, QQ308 enhanced growth of Chinese cabbage. These characteristics were unique among known strains of B. cereus. To our knowledge, this is the first report on the antifungal and Chinese cabbage growth enhancing compounds produced by B. cereus.     

Pathogenesis of Streptoverticillium albireticuli on Caenorhabditis elegans and its antagonism to soil-borne fungal pathogens

AIMS: To examine the biological activity of Streptoverticillium albireticuli. METHODS: Isolation of S. albireticuli was carried out using the dry-heat technique. Nematicidal and pathogenic activity on Caenorhabditis elegans was measured by mortality in metabolites and colonization rate on fishmeal extract agar. Antifungal and enzymatic activities of S. albireticuli were measured by the agar plate method and the semidefined solid media method, respectively. RESULTS: S. albireticuli showed strong nematicidal activity against C. elegans. Pathogenic activity was also evident with the colonized nematode by the isolate on fishmeal extract agar. It also showed antifungal activity against certain fungal pathogens such as Rhizoctonia solani, Phytophthora cinnamomi and Fusarium oxysporum. SIGNIFICANCE AND IMPACT OF THE STUDY: The discovery of an actinomycete showing pathogenic activity against the nematode may indicate the potential for it to be used as a biocontrol agent of parasitic nematodes, in addition to its ability to suppress fungal pathogens.     

Possibilities of the use of vinasses in the control of fungi phytopathogens

The purpose of this research was to study the biocide effect of three agroindustrial subproducts, concretely sugar beet, sugar cane and wine vinasse. Results from in vitro testing determined that wine vinasse is what shows a 100% capacity to suppress fungal growth with concentrations between 5% and 7% for Fusarium oxysporum f.sp. melonis race 0 and 1, Sclerotinia sclerotiorum, Pythium aphanidermatum and Phytophthora parasitica and 10-15% for F. oxysporum f.sp. radicis-cucumerinum. On the other hand, sugar cane vinasse produced an increase at high concentrations and sugar beet vinasse showed an approximate 100% suppressor effect on fungal growth for only some of the phytopathogens tested: S. sclerotiorum (15%), P. aphanidermatum (7%), P. parasitica (15%) and F. oxysporum f.sp. radicis-cucumerinum (15%). In the soil samples analyzed none of the three vinasse extracts decreased fusaric microbiota, producing an increase in the three samples tested. This would implicitly convey an improvement in soil quality by producing a potential increase in bacterial and fungal microbiota.     

Clonostachys rosea BAFC3874 as a Sclerotinia sclerotiorum antagonist: mechanisms involved and potential as a biocontrol agent

Aims: To establish the modes of action of the antagonistic fungal strain Clonostachys rosea BAFC3874 isolated from suppressive soils against Sclerotinia sclerotiorum and to determine its potential as a biocontrol agent. Methods and Results: The antagonistic activity of C. rosea BAFC3874 was determined in vitro by dual cultures. The strain effectively antagonized S. sclerotiorum in pot‐grown lettuce and soybean plants. Antifungal activity assays of C. rosea BAFC3874 grown in culture established that the strain produced antifungal compounds against S. sclerotiorum associated with secondary metabolism. High mycelial growth inhibition coincided with sclerotia production inhibition. The C. rosea strain produced a microheterogeneous mixture of peptides belonging to the peptaibiotic family. Moreover, mycoparasitism activity was observed in the dual culture. Conclusions: Clonostachys rosea strain BAFC3874 was proved to be an effective antagonist against the aggressive soil‐borne pathogen S. sclerotiorum in greenhouse experiments. The main mechanisms involve peptaibiotic metabolite production and mycoparasitism activity. Significance and Impact of the Study: Clonostachys rosea BAFC3874 may be a good fungal biological control agent against S. sclerotiorum. In addition, we were also able to isolate and identify peptaibols, an unusual family of compounds in this genus of fungi.     

Suppressive soil against Sclerotinia sclerotiorum as a source of potential biocontrol agents: selection and evaluation of Clonostachys rosea BAFC1646

The fungal diversity structures of soils that are suppressive and non-suppressive to Sclerotinia sclerotiorum were characterised and screened for fungal strains antagonistic to the S. sclerotiorum pathogen. Soil suppressiveness was associated with a particular fungal diversity structure. Principal component analysis showed that antagonism by fungal species in suppressive soils was associated with the occurrence of Fusarium oxysporum, Fusarium solani, Talaromyces flavus var. flavus and Clonostachys rosea f. rosea. In particular, C. rosea f. rosea occurred exclusively in suppressive soil samples, suggesting that this morpho-species plays an important role in suppression of S. sclerotiorum diseases. One strain of C. rosea f. rosea (BAFC1646) was selected for further experiments. Dual-culture assays confirmed the antagonistic behaviour of C. rosea f. rosea BAFC1646 against three different S. sclerotiorum strains. Antifungal activity was corroborated by diffusion assays with metabolite extracts. Greenhouse assays with soybean plants showed that the selected C. rosea f. rosea strain reduced the percentage of dead plants when co-inoculated with S. sclerotiorum. In addition, inclusion of C. rosea f. rosea alone increased shoot lengths significantly. In this work, we established the involvement of fungal species in soil suppressiveness and in further assays confirmed that C. rosea f. rosea BAFC1646 exhibits a bioprotective effect against S. sclerotiorum in soybean plants.     

Methoxybifurcarenone: an antifungal and antibacterial meroditerpenoid from the brown alga Cystoseira tamariscifolia.

A meroditerpenoid metabolite has been isolated from the brown alga Cystoseira tamariscifolia and characterized as methoxybifurcarenone, by spectral analysis. Methoxybifurcarenone possesses antifungal activity against three tomato pathogenic fungi: Botrytis cinerea, Fusarium oxysporum sp. mycopersici and Verticillium alboatrum and antibacterial activity against Agrobacterium tumefaciens and Escherichia coli.     

离子注入选育高效植物病原真菌拮抗菌JA

Bacillus subtitles JA能够对多种植物病原菌如小麦赤霉菌、西瓜枯萎病菌、黄瓜黑星、油菜菌核、小麦白粉等有良好的抑制效果,本文利用离子注入诱变筛选高效植物病原真菌拮抗菌并且对JA菌株的发酵特性作了进一步的研究,获得了两株高效拮抗菌。其发酵水平的抑菌圈直径从16.62mm提高到21.23mm,经传代实验表明,其遗传性较为稳定。     

Fusion proteins comprising a Fusarium-specific antibody linked to antifungal peptides protect plants against a fungal pathogen

In planta expression of recombinant antibodies recognizing pathogen-specific antigens has been proposed as a strategy for crop protection. We report the expression of fusion proteins comprising a Fusarium-specific recombinant antibody linked to one of three antifungal peptides (AFPs) as a method for protecting plants against fungal diseases. A chicken-derived single-chain antibody specific to antigens displayed on the Fusarium cell surface was isolated from a pooled immunocompetent phage display library. This recombinant antibody inhibited fungal growth in vitro when fused to any of the three AFPs. Expression of the fusion proteins in transgenic Arabidopsis thaliana plants conferred high levels of protection against Fusarium oxysporum f.sp. matthiolae, whereas plants expressing either the fungus-specific antibody or AFPs alone exhibited only moderate resistance. Our results demonstrate that antibody fusion proteins may be used as effective and versatile tools for the protection of crop plants against fungal infection.     

黄瓜转新型抗菌蛋白基因GNK2-1及其抗枯萎病的研究

GNK2-1为一种来自银杏(Ginkgo biloba)种仁的新型抗真菌蛋白, 具有较强的真菌抗性且性质稳定。序列分析表明,其结构与所有已知的抗真菌蛋白不同, 而与富含半胱氨酸的植物类受体激酶的胞外结构域相似。为探索GNK2-1基因在黄瓜(Cucumis sativus)抗病反应中的作用, 利用基因重组技术构建了GNK2-1的高效组成型表达载体, 并利用根癌农杆菌(Agrobacterium tumefaciens)介导转入黄瓜栽培品种农城3号(Cucumis sativus ‘Nongcheng No.3’)基因组中。通过对获得的抗性植株进行PCR、RT-PCR和Western blot检测分析, 结果表明GNK2-1基因可在T₀代转基因植株中转录表达, 并能在T₁代转基因黄瓜中稳定遗传。离体枯萎病抗性鉴定结果表明, 转GNK2-1基因的黄瓜对枯萎病的抗性增强, GNK2-1可以作为黄瓜抗病性改良的潜在基因资源。     

Identification and biocontrol efficacy of Streptomyces miharaensis producing filipin III against Fusarium wilt

A number of bacterial strains were isolated from the internal tissue of Trapa japonica. Of these, strain KPE62302H, which had a 16S rDNA sequence identical to that of Streptomyces miharaensis showed antifungal activity against several plant pathogens. Treatment of seeds with strain KPE62302H induced a significant reduction in the incidence of Fusarium wilt in tomato plants compared with untreated controls. An antifungal substance (FP-1) was purified from the culture extract of strain KPE62302H using C18 flash and Sephadex LH-20 column chromatography and reverse phase HPLC. Extensive spectrometric analysis using MS and NMR identified this as filipin III. FP-1 inhibited the mycelial growth of plant pathogenic fungi such as Alternaria mali, Aspergillus niger, Colletotrichum gloeosporioides, C. orbiculare, Cylindrocarpon destructans, Diaporthe citiri, Fusarium oxysporum at 1-10 μg ml(-1) and also markedly inhibited the development of Fusarium wilt caused by F. oxysporum f.sp. lycopersici in tomato plants by treatment with 10 μg ml(-1) under greenhouse conditions. The efficacy of FP-1 against Fusarium wilt was comparable to that of the synthetic fungicide benomyl. An egfp -tagged strain of KPE62302H confirmed its ability to colonize tomato plants.     

根际真菌对黄瓜土传病害的抑制作用

为了了解根际真菌对黄瓜土传病害的影响,通过平板对峙试验和温室人工接种盆栽试验,对分离自根际土的16株真菌开展了对土传病原真菌的拮抗作用和黄瓜土传病害的抑制作用研究.结果表明: 有4株真菌对一种或多种供试病原真菌表现有拮抗作用,其中经鉴定为土曲霉的菌株JCL143对黄瓜枯萎病菌、立枯病菌和菌核病菌3种供试病原真菌均有较强的拮抗作用.在温室盆栽试验中,接种菌株JCL143对黄瓜3种病害的相对防效均在74%以上;而在不灭菌育苗基质的盆栽试验中,接种菌株JCL143对立枯病和菌核病的防效均在85%以上.在用自然土进行的温室盆栽试验中,接种菌株JCL143对伸蔓期黄瓜枯萎病的相对防效平均达84.1%.菌株JCL143的发酵液对3种供试病原真菌菌落生长都有不同程度的抑制作用,对菌核病菌的菌落生长抑菌率达63.3%.发酵液的抑菌活性随处理温度的升高而下降,对碱性pH值比酸性pH值敏感,而对蛋白酶处理不敏感.说明土曲霉是土壤中抑制植物土传病害的重要因素,菌株JCL143的抑病效果稳定,具有潜在的生防应用价值.