Gloeosporioides Isolates Research Articles

First Report of Resistance to Pyraclostrobin, Boscalid, and Thiophanate-methyl in Colletotrichum gloeosporioides from Blueberry in Georgia

Colletotrichum gloeosporioides causes anthracnose fruit rot and leaf spot on blueberries. For controlling anthracnose, blueberry growers mostly rely on pre- and postharvest fungicide applications in addition to orchard sanitation. Single-site fungicides including quinone outside inhibitors (QoIs), such as pyraclostrobin and azoxystrobin as well as fungicides containing the succinate dehydrogenase inhibitor (SDHI) boscalid are used frequently to control anthracnose rots and other diseases on blueberry; however, development of fungicide resistance is a real risk because a limited number of fungicides are now available for blueberry disease management. In 2019, three isolates of C. gloeosporioides were cultured from blueberry fruit collected from southern highbush blueberry cultivar ‘Farthing’ in two commercial blueberry fields in Pierce County, Georgia, where ripe rot had been a problem. Fungicide sensitivity tests were conducted using a mycelial growth inhibition assay as described previously. A total of nine fungicides were evaluated to determine the sensitivity of these C. gloeosporioides isolates. All three isolates were resistant to thiophanate-methyl, the QoI fungicide pyraclostrobin, and the SDHI fungicide boscalid. These findings suggest that continuous monitoring of fungicide resistance is necessary to avoid the unwarranted application of single-site fungicides.

Comparative pathogenicity of Colletotrichum spp. against different varieties of strawberry plants (Fragaria ananassa) widely grown in Morocco

The evolution of anthracnose symptoms on the aerial part (leaves, stems and strawberries) of three varieties Fortuna, Camarosa and Festival of strawberry plants inoculated with the conidial suspensions of Colletotrichum acutatum and Colletotrichum gloeosporioides isolates was followed. The severity index and infection coefficients increased in function of time. Seven days after inoculation they were low not exceeding 13.43% and 43.33, but they increased four weeks after inoculation, respectively, to 37.96% and 99 on strawberry plants of the Camarosa variety, 54.44% and 105 on those of Fortuna and 51.12% and 85 on those of Festival. At the sixth week, the severity index and infection coefficients became very high, reaching respectively 100% and 408 on Fortuna plants inoculated with C. gloeosporioides isolate (Coll3) followed by Coll2 (89.28% – 300), Coll1 (86.66% – 378) and Coll4 (80.45% – 198) of C. acutatum species. Similarly, the isolate Coll3 caused fruit rot; the percentage of rotten strawberries was 100% on Fortuna variety, 83.33% on Festival and 70.25% on Camarosa. A positive re-isolation of the tested Colletotrichum isolates has been noted from leaves of strawberry varieties and negative from crowns or the roots. A significant to moderate reduction in fresh and dry weights of the aerial part and roots was noted in inoculated strawberry plants compared to the control.

English

Twenty six isolates of Colletotrichum gloeosporioides from anthracnose infected mango fruits were isolated from different places of Tamil Nadu, India and these isolates were identified as C. gloeosporioides by Internal transcribed spacer (ITS) and species specific (CgInt) primers. The sensitivity of C. gloeosporioides isolates to benomyl fungicide were evaluated at five different concentrations viz., 0.5, 1, 2, 5, 10 mg/l through poisoned food technique and were expressed very less to highly sensitiveness to the fungicide. All the twenty six isolates were able to grow at the lowest concentration of 0.5 mg/l benomyl amended medium and the average mycelial growth was 70.70 mm at even days after inoculation. At the highest concentration of 10 mg/l benomyl, only two isolates viz., MCG 7 and 16 were able to grow with the mycelial diameter of 12.00 and 18.00 mm, respectively and 100% inhibition was found in the remaining isolates. In addition, benzimidazole sensitive and resistance β - tubulin gene sequences of TUB 1 and TUB2 were amplified from the benomyl sensitive isolates of C. gloeosporioides. The results indicated the differential resistance or sensitivity to benomyl fungicide against C. gloeosporioides and thereby allowed to identify the variability and diversity of the isolates on regional basis.   Key words: Mango, C. gloeosporioides, benomyl sensitive, β - tubulin gene.

In vitro and in vivo activity of QoI fungicides against Colletotrichum gloeosporioides causing fruit anthracnose in Citrus sinensis

Abstract Anthracnose of sweet orange caused by Colletotrichum gloeosporioides was recently reported as a severe epidemic disease in pre-harvest conditions in several orchards of Sicily, one of the most important orange production areas for Europe. Nowadays, this emerging pathogen strongly reduce the amount of marketable fruits worldwide and fungicide applications are needed. In this study, sensitivity profile to QoI fungicides was preliminarily established for 37 C. gloeosporioides isolates collected from Sicilian orchards and fungal capacity to activate alternative respiration in presence of QoIs, a mechanism known for many phytopathogenic fungi when normal respiration is inhibited, was also determined. Tested isolates showed different fungicide sensitivity levels depending on salicylhydroxamic acid (SHAM) amendment of media. Medians of EC50 values ranged from 0.36 to 102 μg mL–1 for azoxystrobin and from 0.047 to 0.49 μg mL–1 for trifloxystrobin with or without SHAM amendment, respectively. Otherwise, EC50 medians of pyraclostrobin calculated within the same subset of fungal isolates were very similar in presence or in absence of SHAM (0.11 and 0.14 μg mL–1, respectively), thus indicating that only pyraclostrobin among the tested QoIs do not induce alternative respiration for this fungus. Subsequently, four field experiments were carried out in 2013–2014 in the same orange orchards, from which tested isolates were before collected, to determine QoIs performances against C. gloeosporioides. Although under low-medium disease pressure conditions, our data clearly showed that field applications of QoIs significantly managed both the incidence (reductions from 83.7 to 92%) of anthracnose infections on sweet orange in pre-harvest conditions and, approximately reduced up to 81 and 91.6% disease incidence and severity in postharvest stage. Pyraclostrobin, always applied at low rate (about 1/3 of label rate recommended for other targeted fungi of Citrus sinensis) showed good performances in reducing pre- and postharvest anthracnose infections on two cultivars, “Scire” and “Alcala”; of sweet orange. Based on our data, this latter molecule could be considered as a reliable chemical option to counteract this threatening disease on sweet orange, reducing environmental impact and the likelihood of fungicide resistance arising within Italian C. gloeosporioides population.

Most Colletotrichum species associated with tree tomato (Solanum betaceum) and mango (Mangifera indica) crops are not host‐specific

An important constraint for crop production in Colombia is the high incidence of anthracnose caused by Colletotrichum species. Although several studies have focused on these fungi, the relationship between the different fungal species within the genus and their hosts and whether they display any host preference or host specificity has yet to be examined. In Colombia, diseases caused by Colletotrichum species are particularly severe in mango (Mangifera indica) and tree tomato (Solanum betaceum) crops. In a previous investigation, the Colletotrichum phylogenetic species attacking these crops were identified. The present study aimed to determine whether isolates collected from tree tomato and mango showed host preference or host specificity by assessing aggressiveness, spore density, latent period, and fitness of each strain on the two hosts. In the departments of Cundinamarca and Tolima, Colombia, isolates were collected from plants that presented typical anthracnose symptoms and were identified as C. acutatum, C. asianum, C. boninense, C. gloeosporioides, C. tamarilloi and C. theobromicola. Inoculation of conidia of each isolate onto both hosts showed isolates had no host preference and only the C. gloeosporioides isolate showed host specificity. However, in general, isolates produced a higher spore density when inoculated on the alternate host, which may indicate a difference in the degree of adaptation to each host. Statistical analyses of the assessed parameter values revealed that isolates use different infection strategies when infecting each host. In light of these results, the implications of using quantitative estimations of fitness when studying fungal pathogens are discussed.

Sensitivity of Colletotrichum gloeosporioides Isolates from Diseased Avocado Fruits to Selected Fungicides in Kenya

Colletotrichum gloeosporioides is a serious postharvest pathogen of avocado fruits worldwide. Kenya lacks any registered fungicides for the management of the disease. Nevertheless, farmers commonly use commercially available fungicides such as Bayleton 25WP (Triadimefon 250 g/Kg), Milraz 76WP (Propineb 70% and Cymoxanil 6%), and Copper oxychloride 500WP for disease management. The efficacy of these fungicides against C. gloeosporioides is not known. The purpose of this study was therefore to test the inhibitory effect of these fungicides against 46 C. gloeosporioides isolates from avocado fruits collected from varieties grown at different agroecological zones in Murang’a County, a popular avocado-growing region in Kenya. Mycelial growth rate and sporulation for each isolate were measured in vitro on PDA plates amended with different concentrations of the fungicides. Plates were arranged in a completely randomized design with three replications per treatment. All fungicides were effective in vitro but there were significant differences in sensitivity among isolates. Bayleton had the highest mycelial inhibition followed by Milraz, while copper oxychloride had the lowest mycelial inhibition rates, ranging from 81% to 88%. However, copper oxychloride was more effective in inhibiting sporulation. The inhibitory effect of each fungicide was concentration-dependent, where twice the recommended concentration had the highest inhibitory effect, followed by the recommended concentration. Our results show that the fungicides used by farmers against C. gloeosporioides, the causal agent for anthracnose, are effective. We, however, recommend further field tests in different avocado-growing areas so as to validate their efficacy against various isolates and under different environments.

Antifungal activity of five plant-extracted essential oils against anthracnose in papaya fruit

Inhibitory effects of five different plant essential oils were assessed against papaya fruit decay caused by anthracnose. Essential oils were extracted from savory, thyme, cinnamon, mint, and lavender plants and chemical components of each oil were identified by GC-mass spectroscopy. The polymerase chain reaction (PCR) method using the internal transcribed spacer (ITS) primer confirmed the identity of the Colletotrichum gloeosporioides isolate. Then different concentrations of each oil were tested in vitro and in vivo. The in vitro results revealed a 100% reduction of mycelium growth for savory and thyme essential oils. In the in vivo experiment, savory and thyme oils at 2000 μL L−1 caused a 59.26 and 58.40% reduction in lesion diameter and a 64.07 and 54.82% of fruit decay, respectively. Application of savory or thyme oil resulted greater maintenance of fruit firmness than the application of the other essential oils after fruit were inoculated with the anthracnose fungus. Savory oil with the main chemical compound carvacrol (71.2%), and thyme oil with the main chemical constituent thymol (73.3%) were the most effective of the oils tested at controlling anthracnose in vitro and in vivo. The half maximal effective concentration (EC50) of savory oil was the lowest among the extracted oils tested, resulting in EC50 = 58.42 μL L−1 for the in vitro test and EC50 = 1507.19 μL L−1 for the in vivo experiment. This study showed that savory oil is effective as a natural fungicide for controlling anthracnose decay and prolonging the storage life of papaya fruit.

Antifungal effect of poly(lactic acid) films containing thymol and R-(-)-carvone against anthracnose pathogens isolated from avocado and citrus

Biodegradable antifungal films were developed to be used for controlling postharvest anthracnose pathogens. Two antifungal compounds, thymol and R-(-)-carvone, were incorporated into poly(lactic acid) (PLA)-based polymer at 10, 15 and 20% (w/w). Antifungal activity of the pure compounds and the antifungal films against Colletotrichum gloeosporioides isolated from avocado and citrus was evaluated at 12 and 25 °C using vapor diffusion assays. The results indicated that the colony diameter was affected by the vapor concentration of the antifungal compounds in the headspace. At 12 °C, 20% thymol showed complete growth reduction of avocado isolate, while at 25 °C, 15 and 20% thymol showed complete growth reduction of both avocado and citrus isolates. The PLA films incorporated with 15% R-(-)-carvone and 20% thymol were the most effective at 12 °C in suppressing the mycelial growth of the avocado and citrus C. gloeosporioides isolates, respectively, whereas the film incorporated with 20% thymol had the highest antifungal activity against both anthracnose isolates at 25 °C. The inhibitory effect of the antifungal films against anthracnose isolates was correlated to the vapor concentration of the antifungal compounds remaining in the headspace of the Petri dish. Antifungal packaging films can potentially be used to control postharvest pathogens of fresh produce.

Pathogenicity and aggressiveness of Colletotrichum gloeosporioides isolates in ornamental pepper

ABSTRACT Anthracnose, a disease caused by fungi of the Colletotrichum genus, affects many crops, including ornamental plants. This study aimed at evaluating the pathogenicity and aggressiveness of the Colletotrichum gloeosporioides Penz. (Sacc.) isolates Cg1 (papaya), Cg2 (guava) and Cg3 (mango) in the ornamental pepper varieties Stromboli, Etna and Pirâmide, during two different periods (July/August and November/December 2013). The fungi pathogenicity and the severity of plant symptoms were assessed using a grading scale. The three C. gloeosporioides isolates were pathogenic to the three ornamental pepper varieties studied, exhibiting different levels of aggressiveness among them. The environmental conditions influenced the isolates aggressiveness, with the most severe symptoms observed in the second assessment period.

Identification of Colletotrichum species associated with postbloom fruit drop in Brazil through GAPDH sequencing analysis and multiplex PCR

The genus Colletotrichum comprises a group of important fungal pathogens that can infect a wide variety of host plants worldwide. Postbloom fruit drop (PFD) of citrus plants is responsible for extensive crop losses annually, and is particularly detrimental to Brazilian citrus production. The disease was first associated with Colletotrichum gloeosporioides and subsequently linked to Colletotrichum acutatum. However, a new species, C. abscissum, was described in 2015 as the causative agent of PFD in Brazil. The species description used a small number of strains and the distribution of the pathogen remains unclear. The proportion of PFD caused by this species is also unclear, because both C. abscissum and C. gloeosporioides are associated with the disease as well. By analyzing sequences of the GAPDH intron region, we identified 227 isolates of Colletotrichum associated with PFD in orchards of Sao Paulo state, 172 isolates were identified as C. abscissum and 55 as C. gloeosporioides. Morphological characters and multilocus sequencing confirmed species C. abscissum was the only species in the C. acutatum complex associated with PFD disease in Brazil. Although described as sensitive to benzimidazole-based fungicides, 20% of C. gloeosporioides isolates were found in regions with high use of this class of fungicide. Evaluated strains exhibited resistance to this fungicide in vitro. Because previously described primers differentiate between C. acutatum and C. gloeosporioides complexes, but not the particular species associated with PFD, we proposed and validated primers for a single multiplex PCR that specifically distinguished the C. abscissum and C. gloeosporioides sensu stricto.

Variability of Colletotrichum gloeosporioides Isolates the Causal Agent of Anthracnose Disease of Cassava and Yam Plants in Ghana

Cassava and yam plants are important staple foods of millions of people in the tropical world and provide food security in most parts of sub-Saharan Africa. Their production however is affected by anthracnose disease caused by Colletotrichum gloeosporioides, (Penz.) Penz. and Sacc contributing to 30-70% economic loss in both crops. A study was carried out to determine if variations exist between isolates of C. gloeosporioides on the two crops occurring in different agro-ecological zones of Ghana. Anthracnose-diseased samples of cassava and yam plant parts (leaves and stem/vine) were collected from four agro-ecological zones of Ghana (Forest, Forest-Savannah Transition, Guinea and Coastal Savannah). Isolations of the causal organism C. gloeosporioides were made. The isolates were investigated on the basis of growth characteristics on Potato Dextrose Agar (PDA): presence or absence of setae, colour of cultures, dimensions of conidia. Pathogenicity of isolates was determined and molecular analysis conducted using Random Amplified Polymorphic DNA (RAPD). Results showed both similarities and variations in cultural, morphological and growth characteristics of the pathogen. Isolates from yam caused pathogenic lesions on cassava and yam plant sections and vice versa. Forty-four isolates of C. gloeosporioides from cassava and yam compared by RAPD analysis grouped the isolates into three main clusters. Within two of the clusters are six polymorphic sub-groups relating to host and geographical origin. The different polymorphic sub-groups indicated that there are several sub-populations of C. gloeosporioides isolates, which either singly or in association, involved in the yam and cassava anthracnose infections in the field. Isolates from different ecological zones, with similar morphological features, clustering in a polymorphic sub-group suggest a common origin for some of them.

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

AbstractThe 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.

Characterization of Colletotrichum gloeosporioides responsible for anthracnose disease of Trichosanthes kirilowii Maxim in central China

Anthracnose disease is an important fungal disease on Trichosanthes kirilowii Maxim. (T. kirilowii), which has recently become a major problem in production areas of T. kirilowii in Anhui Province, central China. A total of 78 putative Colletotrichum spp. isolates were obtained from infected leaves and fruits of T. kirilowii showing anthracnose symptoms during the years 2009-12. All the fungal isolates were consistently identified as Colletotrichum gloeosporioides by a combination of morphological characteristics, species-specific PCR assays and sequence analysis of the ITS region. The results of pathogenicity tests showed difference in virulence among 24 isolates of C. gloeosporioides representatively selected from different regions, when disease symptoms were evaluated after artificially inoculation on the detached T. kirilowii fruits for 2 weeks. The mycelial growth and germination rate of C. gloeosporioides isolates in vitro were determined under temperatures of 10, 15, 20, 25, 28, 30, 33 and 36°C. The optimum growth temperatures of these isolates were observed at 25-30°C, and the germination rates were rather high in the range of 20-33°C. Host range study revealed that the pathogen, under experimental conditions, can infect nine cultivated and weed species belonging to six families (Cucurbitaceae, Rosaceae, Solanaceae, Convolvulaceae, Celastraceae and Cannabinaceae). Moreover, C. gloeosporioides can overwinter safely in infected tissues in field soil, suggesting the potential contribution of plant debris to disease epidemics in the region. The study presents useful information for epidemiological surveillance of T. kirilowii anthracnose caused by C. gloeosporioides in the central region of China.

Phenotypic and molecular characterization of Colletotrichum species associated with anthracnose of banana (Musa spp) in Malaysia.

Anthracnose caused by Colletotrichum species is a common postharvest disease of banana fruit. We investigated and identified Colletotrichum species associated with anthracnose in several local banana cultivars based on morphological characteristics and sequencing of ITS regions and of the β-tubulin gene. Thirty-eight Colletotrichum isolates were encountered in anthracnose lesions of five local banana cultivars, 'berangan', 'mas', 'awak', 'rastali', and 'nangka'. Based on morphological characteristics, 32 isolates were identified as Colletotrichum gloeosporioides and 6 isolates as C. musae. C. gloeosporioides isolates were divided into two morphotypes, with differences in colony color, shape of the conidia and growth rate. Based on ITS regions and β-tubulin sequences, 35 of the isolates were identified as C. gloeosporioides and only 3 isolates as C. musae; the percentage of similarity from BLAST ranged from 95-100% for ITS regions and 97-100% for β-tubulin. C. gloeosporioides isolates were more prevalent compared to C. musae. This is the first record of C. gloeosporioides associated with banana anthracnose in Malaysia. In a phylogenetic analysis of the combined dataset of ITS regions and β-tubulin using a maximum likelihood method, C. gloeosporioides and C. musae isolates were clearly separated into two groups. We concluded that C. gloeosporioides and C. musae isolates are associated with anthracnose in the local banana cultivars and that C. gloeosporioides is more prevalent than C. musae.

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.

First Report of Colletotrichum acutatum sensu lato Causing Anthracnose on Gooseberry Fruits in the Czech Republic.

Gooseberry (Ribes uva-crispa L.) is a commonly grown fruit tree or bush in the Czech Republic. Colletotrichum acutatum J. H. Simmonds is a polyphagous fungal plant pathogen. This pathogen has been reported causing anthracnose on strawberry in the Czech Republic (2), and recently it has become an important pathogen on the fruits of apple and tomato (4). In 2012, anthracnose symptoms were noticed on fruits of gooseberry (locality Pribyslavice, near Brno). The symptoms on fruit surfaces were round, brown, shriveled, sunken spots of 1.2 to 2.0 cm, with orange conidial masses on the spots. The pathogen was isolated from symptomatic fruits on PDA and cultured at 25 ± 2°C. The color of colonies varied with age from white to gray with occurrence of orange conidial masses. Conidia were colorless and fusiform, size 13 to 17 × 4 to 5 μm (n = 100). The morphological characteristics classified the pathogen as a Colletotrichum sp. To fulfill Koch's postulates, 25 disinfested healthy gooseberry fruits were pinpricked by sterile needle and 10 μl of spore suspension (1 × 105 conidia ml-1) was inoculated by pipetting into the wound. Control fruits were treated with sterile distilled water. The fruits were transferred to a growth cabinet and maintained at a temperature of 25 ± 2°C, relative humidity 70 ± 5%. Similar anthracnose symptoms were observed on all of gooseberry fruits a week after inoculation, whereas no symptoms appeared on control fruits. The pathogen was reisolated from infected fruits. Species determination of the isolates was confirmed by PCR. Specific primers designed in region ITS1, the 5.8S RNA gene, and region ITS2 of the pathogen DNA were selected. Specific primers CaInt2 and ITS4 were used to identify C. acutatum (3), and primers CgInt and ITS4 were used to determine C. gloeosporioides isolate CCM 177 (1), which was used as a control. Our isolates yielded PCR products (size 490 bp) only with primers designed for C. acutatum. The C. gloeosporioides isolate yielded PCR product (size 450 bp) only with CgInt and ITS4 primers. PCR products were sequenced and identified with the BLAST program. The sequence of the gooseberry fruit isolates (Accession No. JX843763 and JX843764) matched with 100% similarity to the C. acutatum sequences in GenBank. To our knowledge, this is the first report of C. acutatum sensu lato on gooseberry fruits in the Czech Republic. This pathogen can endanger the production of gooseberry fruits in this region.

First Report of Colletotrichum gloeosporioides Causing Anthracnose of Banana (Musa spp.) in Malaysia.

Banana is the second largest cultivated fruit crop in Malaysia, and is cultivated for both the domestic market and also for export. Anthranose is a well-known postharvest disease of banana and with high potential for damaging market value, as infection commonly occurs during storage. Anthracnose symptoms were observed on several varieties of banana such as mas, berangan, awak, nangka, and rastali in the states of Perak and Penang between August and October 2011. Approximately 80% of the fruits became infected with initial symptoms characterized as brown to black spots that later became sunken lesions with orange or salmon-colored conidial masses. Infected tissues (5 × 5 mm) were surface sterilized by dipping in 1% sodium hypochlorite (NaOCl) for 3 to 5 min, rinsed with sterile distilled water, and plated onto potato dextrose agar (PDA). Direct isolation was done by transferring the conidia from conidial masses using an inoculation loop and plating onto PDA. For both methods, the PDA plates were incubated at 27 ± 1°C with cycles of 12 h light and 12 h darkness. Visible growth of mycelium was observed after 4 to 5 days of incubation. Twenty isolates with conidial masses were recovered after 7 days of incubation. The isolates produced grayish white to grayish green and grey to moss dark green colony on PDA, pale orange conidial masses, and fusiform to cylindrical and hyaline conidia with an average size of 15 to 19 × 5 to 6 μm. Appresoria were ovate to obovate, dark brown, and 9 to 15 × 7 to 12 μm and setae were present, slightly swollen at the base, with a tapered apex, and brown. The cultural and morphological characteristics of the isolates were similar to those described for C. gleosporioides (1,2,3). All the C. gloeosporioides isolates were deposited in culture collection at Plant Pathology Lab, University Sains Malaysia. For confirmation of the identity of the isolates, ITS regions were sequenced using ITS4 and ITS5 primers. The isolates were deposited in GenBank with accessions JX163228, JX163231, JX163201, JX163230, JX163215, JX163223, JX163219, JX163202, JX163225, JX163222, JX163206, JX163218, JX163208, JX163209, JX163210, JX431560, JX163212, JX163213, JX431540, and JX431562. The resulting sequences showed 99% to 100% similarity with multiple C. gloeosporioides isolates in GenBank. Pathogenicity tests were conducted using mas, berangan, awak, nangka, and rastali bananas. Fruit surfaces were sterilized with 70% ethanol and wounded using a sterile scalpel. Two inoculation techniques were performed separately: mycelia plug and conidial suspension. Mycelial disc (5 mm) and a drop of 20 μl spore suspension (106 conidia/ml) were prepared from 7-day-old culture and placed on the fruit surface. The inoculated fruits were incubated at 27 ± 1°C for 10 days at 96.1% humidity. After 3 to 4 days of inoculation, brown to black spotted lesions were observed and coalesced to become black sunken lesions. Similar anthracnose symptoms were observed on all banana varieties tested. C. gloeosporioides was reisolated from the anthracnose lesions of all the inoculated fruit in which the cultural and morphological characteristics were the same as the original isolates. To our knowledge, this is the first report of C. gloeosporioides causing anthracnose of Musa spp. in Malaysia.

First Report of Stem and Leaf Anthracnose on Blueberry Caused by Colletotrichum gloeosporioides in China.

Blueberry (Vaccinium spp.) is becoming increasingly popular in China as a nutritional berry crop. With the expansion of blueberry production, many diseases have become widespread in different regions of China. In August of 2012, stem and leaf spots symptomatic of anthracnose were sporadically observed on highbush blueberries in a field located in Liaoning, China, where approximately 15% of plants were diseased. Symptoms first appeared as yellow to reddish, irregularly-shaped lesions on leaves and stems. The lesions then expanded, becoming dark brown in the center and surrounded by a reddish halo. Leaf and stem tissues (5 × 5 mm) were cut from the lesion margins and surface-disinfected in 70% ethanol for 30 s, followed by three rinses with sterile water before placing on potato dextrose agar (PDA). Plates were incubated at 28°C. Colonies were initially white, becoming grayish-white to gray with yellow spore masses. Conidia were one-celled, hyaline, and cylindrical with rounded ends, measuring 15.0 to 25.0 × 4.0 to 7.5 μm. No teleomorph was observed. The fungus was tentatively identified as Colletotrichum gloeosporioides (PenZ.) PenZ & Sacc. (teleomorph Glomerella cingulata (Stoneman) Spauld. & H. Schrenk) based on morphological characteristics of the colony and conidia (1). Genomic DNA was extracted from isolate XCG1 and the internal transcribed spacer (ITS) region of the ribosomal DNA (ITS1-5.8S-ITS2) was amplified with primer pairs ITS1 and ITS4. BLAST searches showed 99% identity with C. gloeosporioides isolates in GenBank (Accession No. AF272779). The sequence of isolate XCG1 (C. gloeosporioides) was deposited into GenBank (JX878503). Pathogenicity tests were conducted on 2-year-old potted blueberries, cv. Berkeley. Stems and leaves of 10 potted blueberry plants were wounded with a sterilized needle and sprayed with a suspension of 105 conidia per ml of sterilized water. Five healthy potted plants were inoculated with sterilized water as control. Dark brown lesions surrounded by reddish halos developed on all inoculated leaves and stems after 7 days, and the pathogen was reisolated from lesions of 50% of inoculated plants as described above. The colony and conidial morphology were identical to the original isolate XCG1. No symptoms developed on the control plants. The causal agent of anthracnose on blueberry was identified as C. gloeosporioides on the basis of morphological and molecular characteristics, and its pathogenicity was confirmed with Koch's postulates. Worldwide, it has been reported that blueberry anthracnose might be caused by C. acutatum and C. gloeosporioides (2). However, we did not isolate C. acutatum during this study. To our knowledge, this is the first report of stem and leaf anthracnose of blueberry caused by C. gloeosporioides in China.

Identification and phylogenetic correlation among Colletotrichum gloeosporioides pathogen of anthracnose for mango

Anthracnose is recognized as the most important disease of field and post-harvest disease of mango worldwide. It has affected the fruit production in all countries where mangoes are grown, particularly where high humidity prevails during the cropping session. Severe lesions along with black spots on leaves, inflorescence and fruits on mango (Magnifera indica L.) were observed during routine survey at several sites with mango trees in Eastern Uttar Pradesh indicating the occurrence of anthracnose disease. The pathogen was isolated from several infected samples and matched characteristics of Colletotrichum gloeosporioides Penz. and Sacc. by microscopic, cultural studies and on the basis of spore formation. Pure cultures of C. gloeosporioides were maintained and further identification of this pathogen was conducted using the internal transcribed spacer region of the known C. gloeosporioides rDNA region 5.8S. Sequence similarity of the collected isolates was 100% with some C. gloeosporioides isolates and phylogenetic analysis also showed the close relationship. Although there are many prior reports on anthracnose disease on mango in India, confirmation of this disease on mango from isolates in India is confirmed by sequence analysis for the first time.

First Report of Leaf Spot Disease Caused by Colletotrichum gloeosporioides on Chinese Bean Tree in China.

Chinese bean tree, Catalpa fargesii f. duciouxii (Dode) Gilmour, is an ornamental arbor plant. Its roots, leaves, and flowers have long been used for medicinal purposes in China. During July 2010, severe outbreaks of leaf spot disease on this plant occurred in Kunming, Yunnan Province. The disease incidence was greater than 90%. The symptoms on leaves began as dark brown lesions surrounded by chlorotic halos, and later became larger, round or irregular spots with gray to off-white centers surrounded by dark brown margins. Leaf tissues (3 × 3 mm), cut from the margins of lesions, were surface disinfected in 0.1% HgCl2 solution for 3 min, rinsed three times in sterile water, plated on potato dextrose agar (PDA), and incubated at 28°C. The same fungus was consistently isolated from the diseased leaves. Colonies of white-to-dark gray mycelia formed on PDA, and were slightly brown on the underside of the colony. The hyphae were achromatic, branching, septate, and 4.59 (±1.38) μm in diameter on average. Perithecia were brown to black, globose in shape, and 275.9 to 379.3 × 245.3 to 344.8 μm. Asci that formed after 3 to 4 weeks in culture were eight-spored, clavate to cylindrical. The ascospores were fusiform, slightly curved, unicellular and hyaline, and 13.05 to 24.03 × 10.68 to 16.02 μm. PCR amplification was carried out by utilizing universal rDNA-ITS primer pair ITS4/ITS5 (2). Sequencing of the PCR products of DQ1 (GenBank Accession No. JN165746) revealed 99% similarity (100% coverage) with Colletotrichum gloeosporioides isolates (GenBank Accession No. FJ456938.1, No. EU326190.1, No. DQ682572.1, and No. AY423474.1). Phylogenetic analyses (MEGA 4.1) using the neighbor-joining (NJ) algorithm placed the isolate in a well-supported cluster (>90% bootstrap value based on 1,000 replicates) with other C. gloeosporioides isolates. The pathogen was identified as C. gloeosporioides (Penz.) Penz. & Sacc. (teleomorph Glomerella cingulata (Stoneman) Spauld & H. Schrenk) based on the morphological characteristics and rDNA-ITS sequence analysis (1). To confirm pathogenicity, Koch's postulates were performed on detached leaves of C. fargesii f. duciouxii, inoculated with a solution of 1.0 × 106 conidia per ml. Symptoms similar to the original ones started to appear after 10 days, while untreated leaves remained healthy. The inoculation assay used three leaves for untreated and six leaves for treated. The experiments were repeated once. C. gloeosporioides was consistently reisolated from the diseased tissue. C. gloeosporioides is distributed worldwide causing anthracnose on a wide variety of plants (3). To the best of our knowledge, this is the first report of C. gloeosporioides causing leaf spots on C. fargesii f. duciouxii in China.