Colletotrichum species associated with chili anthracnose in Australia

Phylogenetic relationships were determined for 45 Colletotrichum isolates causing anthracnose disease of chili in Queensland, Australia. Initial screening based on morphology, ITS and TUB2 genes resulted in a subset of 21 isolates being chosen for further taxonomic study. Isolates in the C. acutatum complex were analysed using partial sequences of six gene regions (ITS, GAPDH, ACT, CHS‐1, TUB2 and HIS3), and in the C. gloeosporioides complex were analysed using four gene regions (ITS, TUB2, ApMat and GS). Phylogenetic analysis delineated four Colletotrichum species including C. siamense, C. simmondsii, C. queenslandicum, C. truncatum and a new Colletotrichum species, described here as C. cairnsense sp. nov. This is the first reported association of C. queenslandicum, C. simmondsii and C. siamense with chili anthracnose in Australia; these species were previously associated with anthracnose on papaya and avocado. Furthermore, the dominant species causing anthracnose of chili in Southeast Asia, C. scovillei, was not detected in Australia. Inoculations on chili fruit confirmed the pathogenicity of C. cairnsense and the other four species in the development of chili anthracnose in Australia.     

Phylogenetic and morphological identification of Colletotrichum boninense: a novel causal agent of anthracnose in avocado

In recent years, anthracnose has become a significant disease affecting avocado fruit in the state of Michoacan, Mexico, where it significantly reduces fruit quality and commercial yield. Anthracnose has been assumed to involve Colletotrichum gloeosporioides and C. acutatum as causal agents. However, because of the increasing incidence of anthracnose, a more precise identification of the Colletotrichum spp. involved in this disease has become desirable. During the years 2004–2007, avocado fruits of different sizes exhibiting brown‐black and reddish spots on the pericarp and soft rot in the mesocarp, were gathered from orchards in nine counties. Fungal isolates were cultured on potato dextrose agar, and among these, 31 were selected for molecular, morphological and pathogenicity analyses. The molecular approaches used sequence typing of the internal transcribed spacer region and the partial nuclear large ribosomal subunit, allowing the unequivocal identification of C. gloeosporioides (71%), C. acutatum (16%) and C. boninense (13%). This last species has not been previously reported as being associated with anthracnose symptoms in avocado fruits anywhere in the world. Various morphological characteristics such as the size and shape of conidia were determined, as well as the conidial mass colour. Pathogenicity tests performed with all three species were conducted by inoculating healthy fruits. In each case, identical symptoms developed within 3 days of inoculation. Knowledge of the Colletotrichum populations in the Michoacan state, including the newly encountered avocado pathogen C. boninense, will facilitate further studies addressing the relationships between these Colletotrichum spp. and their avocado host.     

Isolation and pathogenicity of Colletotrichum spp. causing anthracnose of strawberry in south west Spain

Anthracnose, caused by Colletotrichum spp., is a major disease of cultivated strawberry, Fragaria × ananassa. This study identifies the Colletotrichum spp. which causes strawberry anthracnose in the southwest of Spain. A survey of the region was carried out, and the strains isolated were identified as C. acutatum by using the polymerase chain reaction (PCR) with genus and species-specific primers, demonstrating that this species is currently the causal agent of strawberry anthracnose in the studied region. The pathogenicity of C. acutatum and C. gloeosporioides strains was evaluated on two principal strawberry cultivars (cvs Camarosa and Ventana) under field conditions, the latter being more pathogenic than the former.     

Epidemiology,histopathology and aetiology of olive anthracnose caused by Colletotrichum acutatum and C. gloeosporioides in Portugal

Anthracnose is an important disease affecting mature olive fruits, causing significant yield losses, and poor fruit and oil quality. In Portugal, high anthracnose incidence was recorded during 2003–2007 with 41% of 908 orchards surveyed displaying disease symptoms. In another 14% of the orchards, the pathogen was recorded in symptomless plants. Disease severity was on average 36%, frequently reaching 100%. In Portugal, anthracnose is endemic to neglected orchards of susceptible cultivars, but under favourable conditions it can also severely affect less susceptible cultivars. Pathogens were genetically heterogeneous, with Colletotrichum acutatum genetic group A2 as the most frequent (80%), followed by group A4 (12%) and group A5 along with C. gloeosporioides (3–4%), while groups A3 and A6 of C. acutatum were sporadic. Important geographic variations were observed in the frequencies of these populations, accompanied by year‐to‐year populational shifts. Epidemiology and histopathology studies showed the presence of the pathogens on vegetative organs year‐round, particularly on olive leaves and branches, and on weeds. These represent inoculum reservoirs where secondary conidiation occurs, and conidia are then dispersed by spring rains reaching flowers and young fruits or by autumn rains reaching pre‐mature fruits. Unripe fruits were colonized without showing symptoms up to penetration of the cuticle, but further colonization and symptom production was completed only as fruits matured. These findings challenge current control practices, particularly the timing of fungicide treatment, and contribute to improved disease management.     

芒果胶孢炭疽菌漆酶lac1与致病力的相关性分析

为了明确芒果炭疽病病菌(Colletotrichum gloeosporioides)漆酶与该菌侵染相关致病因子之间的关系,本文以愈创木酚为底物筛选获得的高产漆酶菌株A2为材料,测定了漆酶粗提液对芒果炭疽病病菌致病力的影响,并通过半定量RT-PCR法,分析了在不同侵染时段漆酶基因lac1和其他酶基因的表达。结果表明,漆酶粗提液单独不能致病,但能促进该病原菌在寄主中的扩展;在侵染过程中,lac1表达与黑色素合成酶(thd和scd)基因表达相关性更高,与细胞壁降解酶(ecg和pel)基因表达也有一定相关性,但不及前者高。芒果胶孢炭疽菌分泌的漆酶有助于该菌在侵染芒果过程中的扩展,且对侵染相关致病因子黑色素合成酶和细胞壁降解酶基因的表达有一定影响。初步推测,漆酶lac1除了能通过降解芒果组织中的酚类物质、促进病原菌的扩展之外,也可以通过干扰与致病力相关的黑色素合成和细胞壁降解酶的产生来影响芒果胶孢炭疽菌致病力。     

Digital image analysis to measure lesion area of cucumber anthracnose by Colletotrichum orbiculare

A method was developed using scanned computer images and software programs to measure the lesion area on leaves with cucumber anthracnose caused by Colletotrichum orbiculare. After cucumber plants were inoculated with various concentrations of conidia, lesions on diseased leaves were scanned, manipulated with Adobe Photoshop 6.0, and measured using the blob analysis feature in Matrox Inspector 2.2. The method requires relatively low-cost equipment including a scanner, a personal computer, and two programs: Adobe Photoshop 6.0 and Matrox Inspector 2.2. Because stored images are used, the lesion area can be measured as time permits. Processing the images requires about 3 min per sample. The image assessment accurately detected anthracnose lesions on cucumber leaves and could be applied to other foliar necrotic or spot diseases.     

Development of protocols for detection of Colletotrichum acutatum and monitoring of strawberry anthracnose using real-time PCR

Real-time PCR (TaqMan®) assays were developed for the specific detection and discrimination of Colletotrichum spp., C. acutatum and C. gloeosporioides causing anthracnose in strawberry using the most divergent area of the internal transcribed spacers (ITS1 and ITS2) and 5·8S ribosomal RNA (rRNA) gene region. The specificity of the new assays was tested using DNA from six species of Colletotrichum and nine fungal species commonly found associated with strawberry material, and additionally by comparing the sequences with those from databases using a blast search. The sequences only showed identity with homologous sequences from the desired target organisms. The new assays were 10–100 times more sensitive than conventional PCR methods previously published for the diagnosis of strawberry anthracnose. When real-time PCR was compared with ELISA methods, PCR improved the sensitivity of the identification by obtaining positive results for samples of strawberry plant material that tested negative with ELISA. The development of C. acutatum was monitored using artificially infected strawberry crowns from two strawberry cultivars (Camarosa and Ventana) and a real-time PCR assay specific for this species between January and June 2006. The amount of C. acutatum detected using real-time PCR varied significantly by month ( P  < 0·001), but not by cultivar ( P  = 0·394). The new assays were shown to be useful tools for rapid detection and identification of these pathogens and to allow rapid and accurate assessment of the casual agents of anthracnose in strawberry.     

Lack of host specificity of Colletotrichum spp. isolates associated with anthracnose symptoms on mango in Brazil

The aim of the present study was to analyse the genetic and pathogenic variability of Colletotrichum spp. isolates from various organs and cultivars of mango with anthracnose symptoms, collected from different municipalities of São Paulo State, Brazil. Colletotrichum gloeosporioides isolates from symptomless citrus leaves and C. acutatum isolates from citrus flowers with post‐bloom fruit drop symptoms were included as controls. Sequencing of the ITS region allowed the identification of 183 C. gloeosporioides isolates from mango; only one isolate was identified as C. acutatum. amova analysis of ITS sequences showed larger genetic variability among isolates from the same municipality than among those from different populations. fAFLP markers indicated high levels of genetic variability among the C. gloeosporioides isolates from mango and no correlation between genetic variability and isolate source. Only one C. gloeosporioides mango isolate had the same genotype as the C. gloeosporioides isolates from citrus leaves, as determined by ITS sequencing and fAFLP analysis. Pathogenicity tests revealed that C. gloeosporioides and C. acutatum isolates from either mango or citrus can cause anthracnose symptoms on leaves of mango cvs Palmer and Tommy Atkins and blossom blight symptoms in citrus flowers. These outcomes indicate a lack of host specificity of the Colletotrichum species and suggest the possibility of host migration.     

A new anthracnose disease of pyrethrum caused by Colletotrichum tanaceti sp. nov

A new pathogen of pyrethrum (Tanacetum cinerariifolium) causing anthracnose was described as Colletotrichum tanaceti based on morphological characteristics and a four‐gene phylogeny consisting of rDNA‐ITS, β‐tubulin (TUB2), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and actin (ACT) gene sequences. The fungus produced perithecia in culture, requiring an opposite mating type isolate in a heterothallic manner. The initial infection strategy on pyrethrum leaves involved the formation of appressoria followed by production of multilobed infection vesicles in the epidermal cells. Infection and colonization then proceeded through thinner secondary hyphae, which resulted in the initial production of water‐soaked lesions followed by black necrotic lesions. The infection process was suggestive of a hemibiotrophic infection strategy. Moreover, phylogenetic analysis clearly showed that C. destructivum, C. higginsianum and C. panacicola were separate species that also had similar intracellular hemibiotrophic infection strategies as C. tanaceti, which all clustered in the C. destructivum complex. Colletotrichum spp. were detected at 1% incidence in seed of 1 of 19 seed lines, indicating the potential for seed as a source of inoculum into crops. Colletotrichum tanaceti was detected in leaf lesions from 11 of 24 pyrethrum fields surveyed between April and July 2012, at a frequency of 1·3–25·0% of lesions. Anthracnose probably contributes to the complex of foliar diseases reducing green leaf area in pyrethrum fields in Australia.     

河北省小麦主产区禾谷孢囊线虫群体致病型研究

为了明确河北省小麦主产区禾谷孢囊线虫的致病类型,采用27个国际鉴别寄主品种和2个感病小麦品种‘石新733’和‘温麦4’对河北省邯郸、保定、任丘和唐山的4个禾谷孢囊线虫群体致病型进行了鉴定。依据Andersen等鉴别标准,4个群体的致病型不同于国际上已命名的16个禾谷孢囊线虫致病型。唐山群体为一个致病型,它的致病型和山西太谷、安徽固镇群体的致病型相似;邯郸群体、保定群体和任丘群体的致病型属于同一致病型,这3个群体的致病型和定州群体的致病型很接近。对照小麦品种‘石新733’和‘温麦4’对4个禾谷孢囊线虫群体均表现感病。     

Effect of humidity and temperature on conidial germination and appressorium development of two Philippine isolates of the mango anthracnose pathogen Colletotrichum gloeosporioides

A comparison of rates of germination and appressorium formation by an isolate of Colletotrichum gloeosporioides on mango leaves, fruit surfaces and cellophane membranes showed that behaviour was broadly similar on all three substrates. Frequency of appressorium formation was slightly higher on cellophane membranes, and both hyaline and melanized appressoria were formed. Only melanized appressoria were formed on mango surfaces. In vitro experiments on membranes showed comparative differences in physiological behaviour with temperature for two Philippine isolates of C. gloeosporioides . The most stimulatory temperature for production of appressoria differed in isolates I-2 and I-4 (25 and 20°C, respectively). At 30°C more appressoria became melanized than at lower temperatures, but the frequency of formation of penetration pegs was highest at 25°C. Conidia of C. gloeosporioides germinated on cellophane membranes at relative humidities as low as 95%, but the percentage of conidia germinating and forming appressoria increased as the RH approached 100%. Approximately 18% of conidia of C. gloeosporioides I-2 held at 62 and 86% RH for 4 weeks retained viability, and some were capable of forming appressoria when placed at 100% RH. These results have implications for epidemiological models for disease control.     

Anthracnose of Nemesia strumosa Caused by Colletotrichum fuscum

Severe wilt with spots and/or leaf and stem blight were found on a scrophulariaceous flowering plant, Nemesia strumosa, grown in Kagawa Prefecture, Japan, in February 1999. Wilted plants had numerous lesions and died early. A mitosporic fungus isolated repeatedly from the diseased plants was identified as Colletotrichum fuscum and was demonstrated to cause the disease. N. strumosa is a new host for C. fuscum, which has been known to attack foxglove (Digitalis spp.). The present disease was named “anthracnose of N. strumosa” as a new disease. Received 10 October 2000/ Accepted in revised form 11 January 2001     

Importance of different sources of inoculum and dispersal methods of conidia of Colletotrichum musae, the causal agent of banana anthracnose, for fruit contamination

Different populations of Colletotrichum were characterized and quantified on floral parts of banana plants from flowering until harvest. Isolates of Colletotrichum found to be pathogenic and attributed to the species C. musae (77% of isolates) were differentiated from other species by abundant sporulation, a short mycelium, and rapid growth. Colletotrichum musae was isolated from floral parts mainly during the month following bunch emergence. The respective involvement of different sources of inoculum (leaves, bunch bracts, floral parts) in the levels of fruit contamination was evaluated. When the floral parts and bunch bracts were removed at flowering, the severity of anthracnose disease was considerably reduced. The severity of the disease is strongly correlated with cumulative rainfall during the first 35 days after bunch emergence, and was considerably reduced when rainwater runoff over the bunches was limited by placing plastic sleeves over them. The disease was not observed on banana fruit grown under shelters, protected from rain. The results obtained from this study show clearly that contamination of fruit by conidia takes place largely due to the trickling of rainfall over the floral parts, which are the main source of inoculum. The application of these results for integrated control is discussed.     

Morphological and molecular diversity of Colletotrichum spp. causing pepper spot and anthracnose of lychee (Litchi chinensis) in Australia

Since the 1980s a new disease has been affecting Australian lychee. Pepper spot appears as small, black superficial lesions on fruit, leaves, petioles and pedicels and is caused by Colletotrichum gloeosporioides, the same fungus that causes postharvest anthracnose of lychee fruit. The aim of this study was to determine if a new genotype of C. gloeosporioides is responsible for the pepper spot symptom. Morphological assessments, arbitrarily‐primed PCR (ap‐PCR) and DNA sequencing studies did not differentiate isolates of C. gloeosporioides from anthracnose and pepper spot lesions. The ap‐PCR identified 21 different genotypes of C. gloeosporioides, three of which were predominant. A specific genotype identified using ap‐PCR was associated with the production of the teleomorph in culture. Analysis of sequence data of ITS and β‐tubulin regions of representative isolates did not group the lychee isolates into a monophyletic clade; however, given the majority of the isolates were from one of three genotypes found using ap‐PCR, the possibility of a lychee specific group of C. gloeosporioides is discussed.     

Localized hemibiotrophy in Colletotrichum: cytological and molecular taxonomic similarities among C. destructivum, C. linicola and C. truncatum

The infection process of hemibiotrophic isolates of Colletotrichum linicola (from flax, Linum usitatissimum ) and C . truncatum (from broad bean, Vicia faba and lentil, Lens culinaris ) was studied by light microscopy. Host surfaces were penetrated directly leading to a symptomless, biotrophic phase characterized by the elaboration of large multilobed, multiseptate, vesicular primary hyphae that were restricted to the initially infected epidermal cells. Biotrophy lasted for the first 48 h of the host-pathogen interaction and was rapidly succeeded by a necrotrophic phase during which narrow, secondary hyphae invaded the surrounding leaf tissues and water-soaked spreading lesions with sporulating, monosetate acervuli were produced on infected host surfaces. Molecular taxonomic analysis of the nucleotide sequences of the amplified D2 and ITS-2 regions of rDNA revealed very close similarities (97–99%) between these isolates and those of C . destructivum obtained from cowpea ( Vigna unguiculata ) and lucerne ( Medicago sativa ), and also of C . truncatum obtained from pea ( Pisum sativum ). This association was consistent with results from a comparative assessment of some in-planta and in-vitro morphological and growth characteristics of these hemibiotrophic fungi. It was concluded that localized hemibiotrophy is an infection strategy utilized predominantly by a closely-related group of pathogens comprising C . destructivum , C . linicola and C. truncatum , and the formation of multilobed primary hyphae restricted to the first penetrated cell might therefore be a key taxonomic character which correlates consistently with ITS sequence data.     

Variation in the pathogenicity of two Turnip mosaic virus isolates in wild UK Brassica rapa provenances

Brassica rapa can be infected with Turnip mosaic virus (TuMV) as a result of manual inoculation or aphid transmission, but infected plants have not been found in the field. In this study, B. rapa plants grown from seed collected from two field sites in southern England were mechanically inoculated with one of two distinct isolates (pathotypes) of TuMV under glasshouse conditions. These had either been isolated from Brassica oleracea growing wild in Wales, (GBR 83, pathotype 3) or Dorset (GBR 98, pathotype 1). Use of ELISA as an index of infection in manually inoculated B. rapa showed that although seed provenance had a small effect on the proportion of plants infected, the biggest factor was the virus isolate. Both virus isolates infected both lines of B. rapa , but invaded at different rates, although both resulted in easily discernible symptoms. The severity of symptoms was not related to amounts of virus in the infected plants. A significantly greater proportion of plants were infected with GBR 83 at 45 days post-inoculation (d.p.i.) than GBR 98. but GBR 98 caused significantly more severe and obvious symptoms as well as greater mortality at 119 d.p.i., in plants from both sites than GBR 83.     

江苏省水稻品种对水稻细菌性条斑病抗性鉴定及评价

为明确江苏省水稻条斑病菌致病力分化状况和不同类型水稻品种对条斑病抗感特性,在孕穗期采用针刺接种法对徐淮稻区2007—2009年采集分离获得的45株条斑病菌进行致病力测定,根据病原菌在6个水稻鉴别品种IRBB4、IRBB5、IRBB14、IRBB21、IR24和金刚30上的抗感反应划分致病型,从中选择具有代表性的3种不同致病型条斑病菌,并鉴定了240个不同类型的水稻品种对条斑病的抗感性。根据菌株在鉴别品种上的抗感反应将供试菌株划分为8个致病型,其中优势致病型为C3致病型,所占比例为35.5%;大多数菌株与鉴别品种间表现出弱互作关系,少数菌株表现出强互作关系。粳稻品种对条斑病的抗性明显高于籼稻,但常规粳稻和杂交粳稻对强致病力菌抗性比例仅为59.8%和37.5%。表明水稻细菌性条斑病流行仍具有潜在的威胁。     

Aetiology of anthracnose on grapevine shoots in Brazil

Anthracnose is an important disease in vineyards in south and southeast Brazil, the main grape‐producing regions in the country. This study aimed to identify the causal agents of grapevine anthracnose in Brazil through multilocus phylogenetic analyses, morphological characterization and pathogenicity tests. Thirty‐nine Elsinoë ampelina and 13 Colletotrichum spp. isolates were obtained from leaves, stems and berries with anthracnose symptoms collected in 38 vineyards in southern and southeastern Brazil. For E. ampelina isolates, the internal transcribed spacer (ITS), histone H3 (HIS3) and elongation factor 1‐α (TEF) sequences were analysed. HIS3 was the most informative region with 55 polymorphic sites including deletions and substitutions of bases, enabling the grouping of isolates into five haplotypes. Colonies of E. ampelina showed slow growth, variable colouration and a wrinkled texture on potato dextrose agar. Conidia were cylindrical to oblong with rounded ends, hyaline, aseptate, (3.57–) 5.64 (?6.95) μm long and (2.03–) 2.65 (?3.40) μm wide. Seven species of Colletotrichum were identified: C. siamense, C. gloeosporioides, C. fructicola, C. viniferum, C. nymphaeae, C. truncatum and C. cliviae, with a wide variation in colony and conidium morphology. Only E. ampelina caused anthracnose symptoms on leaves, tendrils and stems of Vitis vinifera and V. labrusca. High disease severity and a negative correlation between disease severity and shoot dry weight were observed only when relative humidity was above 95%. In this study, only E. ampelina caused anthracnose symptoms on grapevine shoots in Brazil.     

The spore coat of the bean anthracnose fungus Colletotrichum lindemuthianum is required for adhesion, appressorium development and pathogenicity

The spores (conidia) of the bean anthracnose fungal pathogen, Colletotrichum lindemuthianum, adhere to the aerial parts of plants to initiate the infection process. In previous studies we have shown that the Colletotrichum spores are surrounded by a fibrillar spore coat, comprising several major glycoproteins. Previous evidence showed that a monoclonal antibody (UB20) that recognised these glycoproteins was able to inhibit adhesion of spores to a hydrophobic surface. In this paper we have further studied the role of the spore coat in adhesion, germination and fungal development by studying the effects of UB20 and protease treatment of spores. The latter treatment has previously been shown to remove the spore coat. Spores germinate on glass, polystyrene and water agar, however, appressoria only develop on glass or polystyrene, showing a requirement for a hard surface. Removal of the spore coat with protease inhibits adhesion at 30 min, before the secretion of ECM glycoproteins. Protease treatment also inhibits the development of appressoria and reduces pathogenicity on leaves. Incubation of spores with the MAb UB20 inhibits adhesion at 30 min, but does not affect appressorium formation or pathogenicity. The results suggest that an intact spore coat has two functions; it is required for adhesion to a hydrophobic surface and for the detection of a hard surface necessary for appressorium formation. We suggest that contact with a hard surface, rather than adhesion, is the key event leading to appressorium formation.     

Isolation and pathogenicity of Colletotrichum spp. causing anthracnose of Indian mulberry (Morinda citrifolia) in tropical islands of Andaman and Nicobar, India

This study identifies the Colletotrichum spp. which cause Morinda anthracnose in the Andaman and Nicobar Islands, India. A survey of Indian mulberry plantations was carried out to ascertain the identity of isolates associated with Morinda anthracnose. These were identified as C. gloeosporioides based on morphology and sequencing analysis of internal transcribed spacer (ITS) region, demonstrating that this species is currently the causal agent of Morinda anthracnose in a tropical ecosystem. The pathogenicity test of C. gloeosporioides isolates on Morinda seedlings for 1?month revealed that the five isolates MC1, MC8, MC2 (1), MC4 and MC12 were pathogenic to the respective host with different levels of anthracnose lesion.