Identifying pathogenicity genes in the rubber tree anthracnose fungus Colletotrichum gloeosporioides through random insertional mutagenesis

To gain more insight into the molecular mechanisms of Colletotrichum gloeosporioides pathogenesis, Agrobacterium tumefaciens-mediated transformation (ATMT) was used to identify mutants of C. gloeosporioides impaired in pathogenicity. An ATMT library of 4128 C. gloeosporioides transformants was generated. Transformants were screened for defects in pathogenicity with a detached copper brown leaf assay. 32 mutants showing reproducible pathogenicity defects were obtained. Southern blot analysis showed 60.4% of the transformants had single-site T-DNA integrations. 16 Genomic sequences flanking T-DNA were recovered from mutants by thermal asymmetric interlaced PCR, and were used to isolate the tagged genes from the genome sequence of wild-type C. gloeosporioides by Basic Local Alignment Search Tool searches against the local genome database of the wild-type C. gloeosporioides. One potential pathogenicity genes encoded calcium-translocating P-type ATPase. Six potential pathogenicity genes had no known homologs in filamentous fungi and were likely to be novel fungal virulence factors. Two putative genes encoded Glycosyltransferase family 28 domain-containing protein and Mov34/MPN/PAD-1 family protein, respectively. Five potential pathogenicity genes had putative function matched with putative protein of other Colletotrichum species. Two known C. gloeosporioides pathogenicity genes were also identified, the encoding Glomerella cingulata hard-surface induced protein and C. gloeosporioides regulatory subunit of protein kinase A gene involved in cAMP-dependent PKA signal transduction pathway.     

Actin‐bundling protein fimbrin regulates pathogenicity via organizing F‐actin dynamics during appressorium development in Colletotrichum gloeosporioides

Anthracnose caused by Colletotrichum gloeosporioides leads to serious economic loss to rubber tree yield and other tropical crops. The appressorium, a specialized dome‐shaped infection structure, plays a crucial role in the pathogenesis of C. gloeosporioides. However, the mechanism of how actin cytoskeleton dynamics regulate appressorium formation and penetration remains poorly defined in C. gloeosporioides. In this study, an actin cross‐linking protein fimbrin homologue (CgFim1) was identified in C. gloeosporioides, and the knockout of CgFim1 led to impairment in vegetative growth, conidiation, and pathogenicity. We then investigated the roles of CgFim1 in the dynamic organization of the actin cytoskeleton. We observed that actin patches and cables localized at the apical and subapical regions of the hyphal tip, and showed a disc‐to‐ring dynamic around the pore during appressorium development. CgFim1 showed a similar distribution pattern to the actin cytoskeleton. Moreover, knockout of CgFim1 affected the polarity of the actin cytoskeleton in the hyphal tip and disrupted the actin dynamics and ring structure formation in the appressorium, which prevented polar growth and appressorium development. The CgFim1 mutant also interfered with the septin structure formation. This caused defects in pore wall overlay formation, pore contraction, and the extension of the penetration peg. These results reveal the mechanism by which CgFim1 regulates the growth and pathogenicity of C. gloeosporioides by organizing the actin cytoskeleton.     

胶孢炭疽菌C2H2型转录因子CgGcp1的生物学功能

【背景】胶孢炭疽菌(Colletotrichum gloeosporioides)可以寄生于多种植物,侵染方式多样,能够引起严重的农业危害。在胶孢炭疽菌中,CgGcp1是一个C2H2型的转录因子,关于其生物学功能的研究未见报道。【目的】明确CgGcp1的生物学功能,为深入解析该病菌的致病机制奠定一定的理论依据。【方法】构建CgGCP1基因的敲除载体,利用同源重组得到敲除突变体。通过表型分析,包括营养生长、胁迫响应、孢子产生、附着胞形成及致病性分析等,明确该基因的生物学功能。【结果】CgGCP1基因敲除突变体生长速率较野生型减慢,对SDS、刚果红、NaCl和甘油更加敏感,孢子产量显著降低,附着胞的形成率降低且侵入能力减弱,在橡胶叶片上的致病力明显下降。【结论】CgGcp1参与调控胶孢炭疽菌营养生长、细胞壁完整性、分生孢子产生、附着胞形成与侵入和致病性。     

Physiological characterisation of Colletotrichum gloeosporioides,the incitant of anthracnose disease of noni in India

Ten isolates of Colletotrichum gloeosporioides were collected from different noni growing areas of Tamil Nadu, Karnataka and Kerala in India and their pathogenicity was proved under glass house conditions. Effect of different pH levels, temperature, light intensity and media were tested against the growth of C. gloeosporioides under in vitro. The results indicated that the growth of C. gloeosporioides was maximum in pH range of 6.50–7.00 and temperature range of 25–30°C. Exposure of the fungus to alternate cycles of 12 h light and 12 h darkness resulted in the maximum mycelial growth of C. gloeosporioides compared to the 24 h exposure to continuous light and 24 h exposure to continuous dark. Among the different media tested, host leaf extract medium supported significantly the maximum growth of all the 10 isolates of C. gloeosporioides followed by potato dextrose agar. Further, the strains were found to vary morphologically between the isolates under the study.     

Isolation and Molecular Identification of Colletotrichum gloeosporioides Causing Brown Spot Disease of Camellia oleifera in Hainan of China

Colletotrichum gloeosporioides is an important pathogen that causes widespread brown spot disease on the leaves of the tea‐oil tree (Camellia oleifera) in China. This study was designed to isolate, identify and characterize this fungal pathogen, based on morphology, molecular characteristics and pathogenicity. One pathogenic fungus, named CCG4, was isolated from wild‐type Camellia oleifera of Hainan Province. Colonies were regular circular in shape with 50–60 mm diameter after 5 days of incubation at 28°C on potato dextrose agar (PDA) medium, and woolly with a small amount of jacinth pigment; the colony colour changed from white to black during later stages of infection. The mycelium produced was branched and septate. Conidia were cylindrical‐truncate, oblong‐obtuse to doliform, colourless with 1–2 hyaline oil globules and 4.5–5.3 μm × 7.7–17.5 μm. The sporodochia were cushion‐shaped. The pathogen was identified as Colletotrichum gloeosporioides on the basis of morphological characteristics and internal transcribed spacer sequence, which showed 100% query cover and 99% similarity to the sequence Colletotrichum gloeosporioides JN887341.1 , from a pathogenic fungus known to cause brown spot disease of Camellia oleifera.     

Molecular characterization and pathogenicity of Colletotrichum sp. from guava

Guava (Psidium guajava) fruit is vulnerable to postharvest diseases, such as anthracnose. In the present study, molecular characterisation and pathogenicity of Colletotrichum associated with antharcnose disease of guava fruit were conducted. From anthracnose lesion of guava, 20 isolates were successfully recovered. Based on colony colours, conidia, appressoria and presence or absence of setae, and ITS regions and ß-tubulin gene sequences, the isolates were identified as Colletotrichum gloeosporioides. Phylogenetic analysis based on combined data-sets using neighbour-joining method showed that C. gloeosporioides isolates did not group with C. gloeosporioides epitype strain, and thus the isolates were referred to as C. gloeosporioides species complex or C. gloeosporioides sensu lato. Pathogenicity tests using wounded treatment showed that C. gloeosporioides isolates from guava were pathogenic causing anthracnose on the fruits. The present study showed that C. gloeosporioides sensu lato is the most common species causing antharcnose disease of guava fruit.     

Carbamoyl phosphate synthetase subunit Cpa1 interacting with Dut1, controls development,arginine biosynthesis,and pathogenicity of Colletotrichum gloeosporioides

Carbamoyl phosphate synthetase is involved in arginine biosynthesis in many organisms. In this study, we investigate the biological function of Cpa1, a small subunit of carbamoyl phosphate synthetase of Colletotrichum gloeosporioides. The deletion of the CPA1 gene affected vegetative growth, arginine biosynthesis, and fungal pathogenicity. Genetic complementation with native CPA1 fully recovered all these defective phenotypes. We observed that Cpa1-RFP fusion protein is localized at the mitochondria, which is consistent with Cpa2, a large subunit of carbamoyl phosphate synthetase. We identified the proteins that interact with Cpa1 by using the two-hybrid screen approach, and we showed that Dut1 interacts with Cpa1 but without Cpa2 in vivo. Dut1 is dispensable for hyphal growth, appressorial formation, and fungal pathogenicity. Interestingly, the Dut1-Cpa1 complex is localized at the mitochondria. Further studies showed that Dut1 regulates Cpa1-Cpa2 interaction in response to arginine. In summary, our studies provide new insights into how Cpa1 interacts with its partner proteins to mediate arginine synthesis.     

Lack of Involvement of Nutrients in the Latency of Colletotrichum gloeosporioides in Unripe Avocado Fruits

Hypobaric pressures prevented the ripening of avocado fruit discs and the development of Colletotrichum gloeosporioides infection on them. The same condition did not prevent the growth of C. gloeosporioides on potato dextrose agar. Using this system it was shown that the resistance of unripe avocado fruit discs does not originate in the lack of subsrate for fungal growth.     

In Vitro Evaluation of Antagonism of Endophytic Colletotrichum gloeosporioides Against Potent Fungal Pathogens of Camellia sinensis

An endophytic fungus isolated from Camellia sinensis, Assam, Northeastern India was identified as Colletotrichum gloeosporioides on the basis of morphological characteristics and rDNA ITS analysis. This endophytic fungus was evaluated for growth inhibition against tea pathogens Pestalotiopsis theae and Colletotrichum camelliae. One isolate of C. gloeosporioides showed strong antagonistic activity against Pestalotiopsis theae (64 %) and moderate activity against C. camelliae (37 %). Fifty percent cell-free culture filtrate from 5-day-old cultures showed highest antagonistic activity against both the pathogens although the inhibition percent was less as compared to dual culture. In the experiment of volatile compounds none of the isolates of C. gloeosporioides strains showed visible inhibition against P. theae and C. camelliae. The activity of extracellular hydrolytic enzymes chitinase and protease was also high in this culture fluid and measured 10 and 4.3 IU/μl, respectively.     

First Report of Anthracnose Disease on Jatropha curcas Caused by Colletotrichum gloeosporioides in Korea

In the summers of 2010 and 2011, an anthracnose disease was observed on the Jatropha curcas L. grown at the research field of Gyeongsangnam‐do Agricultural Research and Extension Services, South Korea. The symptoms included the appearance of dark brown spots on the leaf and fruit and the mummification of the fruit. The causal fungus formed grey to dark grey colony on potato dextrose agar. Conidia were single celled, ovoid or oblong, and 8–15 × 3–5 μm in size while seta was dark brown, cone‐shaped and 25–46 × 2–6 μm in size. The optimum temperature for growth was approximately 30°C. On the basis of mycological characteristics, pathogenicity test and molecular identification using internal transcribed spacer rDNA sequence, the fungus was identified as Colletotrichum gloeosporioides. To our knowledge, this is the first report of an anthracnose caused by C. gloeosporioides on J. curcas plant in Korea.     

The CsSTE50 Adaptor Protein in Mitogen-Activated Protein Kinase Cascades Is Essential for Pepper Anthracnose Disease of Colletotrichum scovillei

Anthracnose, caused by the ascomycete fungus Colletotrichum scovillei, is a destructive disease in pepper. The fungus germinates and develops an infection structure called an appressorium on the plant surface. Several signaling cascades, including cAMP-mediated signaling and mitogen-activated protein kinase (MAPK) cascades, are involved in fungal development and pathogenicity in plant pathogenic fungi, but this has not been well studied in the fruit-infecting fungus C. scovillei. Ste50 is an adaptor protein interacting with multiple upstream components to activate the MAPK cascades. Here, we characterized the CsSTE50 gene of C. scovillei, a homolog of Magnaporthe oryzae MST50 that functions in MAPK cascades, by gene knockout. The knockout mutant ΔCsste50 had pleiotropic phenotypes in development and pathogenicity. Compared with the wild-type, the mutants grew faster and produced more conidia on regular agar but were more sensitive to osmotic stress. On artificial and plant surfaces, the conidia of the mutant showed significantly reduced germination and failed to form appressoria. The mutant was completely non-pathogenic on pepper fruits with or without wounds, indicating that pre-penetration and invasive growth were both defective in the mutant. Our results show that the adaptor protein CsSTE50 plays a role in vegetative growth, conidiation, germination, appressorium formation, and pathogenicity in C. scovillei.     

The Colletotrichum gloeosporioides perilipin homologue CAP 20 regulates functional appressorial formation and fungal virulence

Previous studies of the CAP20 gene in Colletotrichum gloeosporioides show that the CAP20 gene may affect virulence in avocados and tomatoes. In this study, we characterized the function of CAP20 from C. gloeosporioides, the causal agent of Colletotrichum leaf fall disease of Hevea brasilience. CAP20 encodes a perilipin homologue protein. Further investigations showed that the Cap20‐GFP fusion protein localized in lipid droplets in hypha and conidia. A C. gloeosporioides mutant, lacking CAP20, had thinner spores and smaller appressoria, and its turgor pressure generation was dramatically reduced and pore size was enlarged. Furthermore, we tested the pathogenicity of conidia from the wild type, gene‐deleted mutant and complemented transformant C.gloeosporioides on the leaves of rubber trees in sterile water and 0.19 M PEG2000. Conidia from the wild type and complemented transformant C. gloeosporioides in 0.19 M PEG2000 caused necrotic lesions and did not produce any lesion with the CAP20 null mutant. But all of them had developed normal disease lesions when they were inoculated in water. These results suggest that CAP20 is a perilipin homologue protein and is involved in functional appressoria development in C. gloeosporioides. CAP20 gene only affects fungal virulence to some extent by reducing the penetration of the immature appressoria into host cuticle in C. gloeosporioides.     

蛋白-O-岩藻糖基转移酶1基因CfPOFUT1在果生炭疽菌中的功能分析

【目的】蛋白-O-岩藻糖基转移酶1 (protein O-fucosyltransferase 1,POFUT1)是催化蛋白质O-岩藻糖基化的关键酶,在动物和人体内被证明调控一系列的生理病理过程,然而POFUT1基因在果生炭疽菌乃至真菌中还未见报道。本研究旨在克隆果生炭疽菌中CfPOFUT1基因,并分析其生物学功能。【方法】利用RT-PCR技术扩增CfPOFUT1的基因并进行生物信息学分析,构建了CfPOFUT1基因的沉默和过表达载体,通过PEG介导法将载体导入原生质体中获得CfPOFUT1基因的沉默和过表达突变体。测定了野生型菌株、CfPOFUT1沉默菌株和过表达菌株在PDA上的菌丝生长、分生孢子产生、萌发与附着胞形成、胁迫应答和致病力、杀菌剂敏感性等生物学表型。【结果】与野生型菌株相比,基因过表达突变体产孢量显著增加,致病力增强,对嘧菌酯敏感性降低,但对多菌灵和咪鲜胺敏感性增强。基因沉默突变体产孢量减少,细胞壁完整性、内质网应激敏感性提高,致病力减弱,对嘧菌酯敏感性提高,但对多菌灵和咪鲜胺敏感性降低。【结论】CfPOFUT1基因参与调控果生炭疽菌分生孢子产量,细胞壁完整性、内质网对应激和药剂敏感性,并对其致病性也具有一定的影响。     

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.     

胶孢炭疽菌CgRGS2基因的克隆及生物学功能

【目的】G蛋白信号调控因子(Regulators of G-protein signaling,RGS)是G蛋白的一类负调控因子,在植物病原菌生长发育及致病过程中发挥着重要的作用,然而目前还未有关于胶孢炭疽菌RGS蛋白生物学功能的研究。本试验的目的是克隆胶孢炭疽菌的一个RGS基因CgRGS2,并分析其生物学功能。【方法】利用PCR技术扩增CgRGS2的基因并进行生物信息学分析,利用同源重组的方法获得CgRGS2基因的敲除突变体,并在突变体的基础上获得互补株,通过表型分析确定该基因的生物学功能。【结果】通过PCR扩增获得了CgRGS2的基因,其编码一个574个氨基酸的蛋白,在N末端含有一个RGS功能域。该基因敲除突变体同野生型相比,表现为营养生长缓慢,气生菌丝浓密,分生孢子产量降低且孢子呈多端萌发,对氧化压力及SDS敏感,致病性减弱等。【结论】CgRGS2蛋白参与调控胶孢炭疽菌的营养生长,分生孢子产量及萌发,氧化应激反应及细胞壁完整性,对其致病性也具有一定的影响。     

CtPMK1, a mitogen‐activated‐protein kinase gene,is required for conidiation,appressorium formation,and pathogenicity of Colletotrichum truncatum on soybean

Colletotrichum truncatum, the causal agent of soybean anthracnose, invades host plants by forming a specialised infection structure called an appressorium. Mitogen‐activated protein kinase (MAPK) genes have been shown to play vital roles in several phytopathogenic fungi in regulating various infection processes, including spore germination, melanised appressorium formation, appressorial penetration and subsequent invasive growth in host plants. In this study, we identified and characterised the first Fus3/Kss1‐related MAPK gene, CtPMK1, in Colletotrichum truncatum, which is related to PMK1 in Magnaporthe oryzae. Disruption of CtPMK1 in C. truncatum resulted in a mutant with slightly reduced mycelial growth (‐30%) and melanisation that is deficient in sporulation (‐99%), as observed in the CMK1 mutant of Colletotrichum lagenarium (a synonym of Colletotrichum orbiculare, which is now the accepted name for this taxon). In contrast to CMK1 of C. lagenarium, conidia from the Ctpmk1 mutant germinated normally on glass slides and onion epidermal surfaces. Our findings suggest that there are differences in the types of in vitro functions controlled by PMK1, even between closely related species. Furthermore, the Ctpmk1 mutant failed to form appressoria or hyphopodia, subsequently resulting in the complete loss of pathogenicity on host plants. Overall, the results indicate that the Fus3/Kss1‐related MAPK gene has a conserved role in infection structure formation and pathogenicity in phytopathogenic fungi.