The Colletotrichum graminicola striatin orthologue Str1 is necessary for anastomosis and is a virulence factor

Striatin family proteins are key regulators in signalling pathways in fungi and animals. These scaffold proteins contain four conserved domains: a caveolin‐binding domain, a coiled‐coil motif and a calmodulin‐binding domain at the N‐terminus, and a WD‐repeat domain at the C‐terminus. Fungal striatin orthologues are associated with sexual development, hyphal growth and plant pathogenesis. In Fusarium verticillioides, the striatin orthologue Fsr1 promotes virulence in the maize stalk. The relationship between fungal striatins and pathogenicity remains largely unexplored. In this study, we demonstrate that the Colletotrichum graminicola striatin orthologue Str1 is required for full stalk rot and leaf blight virulence in maize. Pathogenicity assays show that the striatin mutant strain (Δstr1) produces functional appressoria, but infection and colonization are attenuated. Additional phenotypes of the Δstr1 mutant include reduced radial growth and compromised hyphal fusion. In comparison with the wild‐type, Δstr1 also shows a defect in sexual development and produces fewer and shorter conidia. Together with the fact that F. verticillioides fsr1 can complement Δstr1, our results indicate that C. graminicola Str1 shares five phenotypes with striatin orthologues in other fungal species: hyphal growth, hyphal fusion, conidiation, sexual development and virulence. We propose that fungal striatins, like mammalian striatins, act as scaffolding molecules that cross‐link multiple signal transduction pathways.     

Immunoassay‐based Techniques for Early and Specific Detection of Latent Postharvest Anthracnose in Mango

The postharvest anthracnose pathogen Colletotrichum gloeosporioides inciting latent or quiescent infection of mango was detected in early stages using immunoassay methods. Twenty‐five pathotypes isolated from different agroclimatic zones of Tamil Nadu, Karnataka and Pondicherry, India, revealed the variation in protein profile analysis (SDS‐PAGE). The polyclonal antibodies (PCA) were raised against the unfractioned mycelial protein (UMP) and a 40‐kDa polypeptide present in all pathotypes. Standardization of antigen and antiserum dilutions revealed that an antigen dilution of 1 : 200 (protein concentration of 20 μg/ml) and antiserum dilution of 1 : 100 (protein concentration of 40 μg/ml raised against UMP) and 1 : 200 (protein concentration of 20 μg/ml raised against 40 kDa polypeptide) was found to be optimum for the detection of anthracnose pathogen. Both antisera detected the C. gloeosporioides antigen in enzyme‐linked immunosorbent assays (ELISAs), dot immunobinding assays (DIBAs) and Western blots. The specificity in reaction was compared by isolating other Colletotrichum spp. from various hosts viz., C. lindemuthianum (beans), C. falcatum (sugarcane), C. musae (banana), C. capsici (chillies) and Botryodiplodia theobromae (mango). The antisera generated against UMP revealed the cross‐reaction with other host isolates and mango stem end rot pathogen (B. theobromae). The PCA raised against 40‐kDa polypeptide exhibited the specific reaction with C. gloeosporioides isolates in all the immunoassay techniques. By utilizing both PCA, the presence of latent infection was observed in healthy‐looking leaves, flowers and fruits in orchard conditions. The fruit tissues recorded high absorbance values followed by flowers and leaves in all the detection methods. The ELISA technique was also useful in assessing the pathogen inoculum at various biocontrol formulations sprayed mango trees under field conditions. The fluorescent pseudomonad strains mixture (KFP1 + FP7) amended with chitin sprayed at 30‐day intervals revealed the significant reduction in pathogen load than other formulations and unsprayed control.     

纳他霉素对芒果采后胶孢炭疽菌的抑菌效果及机理

以纳他霉素为抑菌剂, 实验测定了离体条件下不同浓度纳他霉素对胶孢炭疽菌(Colletotrichum gloeosporioides)的孢子萌发及菌丝生长的抑制效果, 以及活体损伤接种炭疽病菌后, 纳他霉素对芒果(Mangifera indica)果实炭疽病的防治效果。通过测定纳他霉素处理后胶孢炭疽菌的细胞膜相对渗透率、可溶性蛋白含量、细胞膜完整性、孢子内活性氧水平和线粒体分布情况, 初步探明其抑菌机理。结果表明, 3 mg∙L ^-1纳他霉素可显著抑制胶孢炭疽菌孢子萌发、芽管伸长和菌落生长, 80 mg∙L ^-1纳他霉素可有效抑制芒果贮存过程中果实炭疽病斑的扩展。纳他霉素处理后胶孢炭疽菌细胞膜相对渗透率和可溶性蛋白含量增加; 2 mg∙L ^-1纳他霉素处理8小时, 处理组胶孢炭疽菌孢子细胞膜损伤染色率为33.6%, 对照组染色率为13.9%; 处理组胞内活性氧产生染色率达46.9%, 比对照组高39.7%; 同时观察到纳他霉素使胞内线粒体分布不均且荧光信号微弱。以上结果表明, 纳他霉素可以破坏胶孢炭疽病菌细胞膜, 诱导活性氧大量积累, 并降低线粒体活性, 从而干扰菌体正常生理活性, 使其代谢活动受影响, 从而达到抑菌目的。     

基于Cfku70基因失活策略构建果生刺盘孢的高效基因敲除底盘菌株

果生刺盘孢侵染危害多种植物,是重要的植物病原真菌。在一些丝状真菌中,敲除非同源末端连接修复通路的关键基因ku70或ku80可显著提高同源重组效率,进而提高靶基因置换频率。本研究从果生刺盘孢基因组鉴定到Cfku70和Cfku80两个基因,并明确了基因失活对菌株生物学表型和基因敲除效率的影响。敲除Cfku70或Cfku80不影响菌株的菌落形态、营养生长、产孢、分生孢子萌发、侵染结构发育和致病;Cfku70基因敲除还大幅提升3个测试基因的敲除效率。本研究证实Cfku70基因失活能显著提高果生刺盘孢的基因敲除效率,适宜作为高效基因敲除的底盘菌株,研究结果为通过批量敲除策略筛选新型致病因子奠定重要基础。     

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.     

A new class of tetraspanins in fungi

Tetraspanins are animal proteins involved in membrane complexes that are involved in cell adhesion, differentiation, and motility. The PLS1 gene from rice blast fungus Magnaporthe grisea encodes a protein (Pls1p) structurally related to tetraspanins that is required for pathogenicity. In Botrytis cinerea public sequences, we identified an EST homologous to PLS1. Using degenerated oligonucleotides, we amplified sequences homologous to PLS1 in fungi Colletotrichum lindemuthianum and Neurospora crassa. Analysis of N. crassa and M. grisea genome sequences revealed the presence of a single tetraspanin gene. Thus, fungi differ from animals, which contain between 20 and 37 paralogous tetraspanin genes. Fungal proteins encoded by BcPLS1, ClPLS1, and NcPLS1 display all the structural hallmarks of tetraspanins (predicted topology with four transmembrane domains, extra- and intracellular loops; conserved cysteine-based patterns in second extracellular loop). Phylogenetic analysis suggests that these genes define a new family of orthologous genes encoding fungal-specific tetraspanins.     

Mycolytic effect of extracellular enzymes of antagonistic microbes to Colletotrichum falcatum,red rot pathogen of sugarcane

Strains of selected bacteria and Trichoderma harzianum isolated from sugarcane rhizosphere and endosphere regions were tested for the production of chitinolytic enzymes and their involvement in the suppression of Colletotrichum falcatum, red rot pathogen of sugarcane. Among several strains tested for chitinolytic activity, 12 strains showed a clearing zone on chitin-amended agar medium. Among these, bacterial strains AFG2, AFG 4, AFG 10, FP7 and VPT4 and all the tested T. harzianum strains produced clearing zones of a size larger than 10 mm. The antifungal activity of these strains increased when chitin was incorporated into the medium. Trichoderma harzianum strain T5 showed increased levels of activity of N-acetylglucosaminidase and -1,3-glucanase when grown on minimal medium containing chitin or cell wall of the pathogen. Lytic enzymes of bacterial strains AFG2, AFG4, VPT4 and FP7 and T. harzianum T5 inhibited conidial germination and mycelial growth of the pathogen. Enzymes from T. harzianum T5 were found to be the most effective in inhibiting the fungus. When mycelial discs of the pathogen were treated with the enzymes, electrolytes were released from fungal mycelia. The results indicated that antagonistic T. harzianum T5 caused a higher level of lysis of the pathogen mycelium, and the inhibitory effect was more pronounced when the lytic enzymes were produced using chitin or cell wall of the pathogen as carbon source.     

西瓜炭疽病菌圆刺盘孢分生孢子产生条件的研究

为研究圆刺盘孢Colletotrichum orbiculare的分生孢子产生条件,通过改变培养基的成分、理化性状及培养条件等方法,进行了诱导产孢试验,并对所产生的分生孢子量进行测定。结果表明,C.orbiculare在西瓜茎叶煎汁培养基上25℃恒温黑暗培养30d可产生大量分生孢子,产孢量高达7.15×109个/m L。在显微镜下观察发现,病菌在西瓜茎叶煎汁培养基上和在西瓜茎秆病斑上产生的分生孢子在形态上存在差异,前者产生的分生孢子稍短和略宽,两端钝圆;而后者产生的分生孢子相对长而细,两端较尖。试验证明寄主西瓜茎叶成分对病菌分生孢子的产生具有明显促进作用。     

珍珠李炭疽菌对三种杀菌剂的敏感性测定

由胶孢炭疽菌复合种(Colletotrichum gloeosporioides species complex)引起的炭疽病是珍珠李(Prunus salicina Lind1)产业生产上的主要病害，为明确珍珠李炭疽菌对吡唑醚菌酯、苯醚甲环唑和咪鲜胺的敏感性，比较不同地理来源及不同种炭疽菌对三种药剂敏感性差异。采用菌丝生长速率法分别测定三种杀菌剂对采自不同地区26株珍珠李炭疽菌的药剂敏感性。结果表明，供试的26株珍珠李炭疽菌对咪鲜胺的敏感性最高，EC₅₀值介于0.010 8～0.645 2μg/mL之间，平均值为(0.191 2±0.03)μg/mL;苯醚甲环唑次之，EC₅₀值介于0.165 8～2.168 0μg/mL之间，平均值为(0.725 8±0.088 0)μg/mL;吡唑醚菌酯最低，EC₅₀值介于0.058 3～84.566 0μg/mL之间，平均值为(6.276 3±3.525 5)μg/mL。供试的26株珍珠李炭疽菌整体对以上三种杀菌剂敏感，且对咪鲜胺的敏感性最高，苯醚甲环唑次之，对吡唑醚菌酯的敏感性最...     

Effect of row spacing on biological control of sicklepod (Senna obtusifolia) with Colletotrichum gloeosporioides

In field experiments conducted over 3 years, the mycoherbicidal fungus Colletotrichum gloeosporioides, formulated either in 20% (v/v) unrefined corn oil and 0.2% Silwet L-77 surfactant or with an invert emulsion, provided season-long control of Senna obtusifolia in narrow (51 cm) rows of soybean. However, in wide (102 cm) rows, one application of either formulation failed to provide season-long control of S. obtusifolia, and two applications were required to achieve season-long weed control. In narrow (51 cm) rows, one application of the fungus either in unrefined corn oil or an invert emulsion controlled S. obtusifolia an average of >90%, and a second application was not required for season long weed control. Soybean yields in wide-row plots treated with two applications of either the fungus/corn oil or fungus/invert emulsion, or with a single application of the fungal treatments in narrow-row soybean plots, were not significantly different from weed-free control plots, or from plots treated with the herbicide chlorimuron. These results suggest that row spacing can affect mycoherbicidal efficacy of this fungus for controlling S. obtusifolia.