Gloeosporioides Isolates Research Articles

Morphological and Molecular Diversity of Colletotrichum gloeosporioides Causing Yam (Dioscorea alata L.) Anthracnose Disease in Côte d’Ivoire

Yam is a plant of great importance for the Ivorian people in terms of food, nutrition, economy, and socio-cultural value. However, its production is threatened by several biotic factors, including anthracnose, one of the most destructive diseases affecting yams. The objective of this study was to assess the morphological and molecular diversity of Colletotrichum gloeosporioides isolates responsible for anthracnose in yam (Dioscorea alata) in Côte d'Ivoire. Twenty-seven previously isolated strains were collected from Plant Health Laboratory of Nangui ABROGOUA University and analyzed. The study examined the cultural (coloration, growth mode, texture, and colony diameter), microscopic (conidial shape, size, and spore concentration), and molecular characteristics of these isolates. Significant morphological diversity was observed among the isolates. Macroscopically, three major groups were identified based on colony coloration (whitish, orange, and grayish). Colony textures varied, presenting cottony, flaky, downy, or filamentous appearances, with growth patterns ranging from concentric and radiating to regular rings. Colony diameters ranged from 37 mm to 82 mm. Microscopically, sporulation varied significantly among isolates, ranging from 1.1 × 10⁵ to 85.4 × 10⁵ spores, as did conidial size, which ranged from 10.54 to 19.29 µm in length and 3.93 to 6.43 µm in diameter. Molecular analysis through comparison with GenBank nucleotide sequences revealed homology rates of 99-100% and varying proximities between the rDNA sequences of the isolates. In conclusion, the characterization of these isolates provides valuable insights for improving disease control strategies for yam anthracnose.

Resistant risk and resistance mechanism of florylpicoxamid in Colletotrichum gloeosporioides isolated from Chinese walnut

Colletotrichum gloeosporioides is the causal pathogen for the devastating walnuts anthracnose. A novel quinone inside inhibitor (QiI) fungicide florylpicoxamid has strong inhibitory efficacy against C. gloeosporioides. This study looked into the resistance risk and mechanism of C. gloeosporioides to florylpicoxamid. The basal level sensitivity of C. gloeosporioides isolates (n = 102) to florylpicoxamid was established with an average 50% mycelial growth inhibition concentration (EC50) value of 0.069 ± 0.035 μg/mL. Six stable florylpicoxamid-resistant mutants with resistance factors of >1000 were produced. The fitness of every mutant was much lower than that of their parental isolates. In general, the resistance risk of C. gloeosporioides to florylpicoxamid would be moderate. Molecular docking results revealed that the amino acid substitutions A37V, and S207L in CgCytb lead to a reduction in the binding affinity between florylpicoxamid and CgCytb, indicating that these two mutations (S207L and A37V in CgCytb) indeed confer florylpicoxamid resistance in C. gloeosporioides. These findings offer a fresh viewpoint on the mechanism underlying QiI fungicide resistance and could support the prudent application of florylpicoxamid in the future to combat walnut anthracnose.

Molecular Characterization of Colletotrichum gloeosporioides (Glomerella cingulata) Causing Mango Anthracnose Disease

Mango production in Tamil Nadu is known to be adversely affected by C. gloeosporioides in both field and post harvest stage of crop and drastically reduces market and edible value of fruit. To manage the disease it is essential to learn about the nature of pathogen and its way of interaction with host and its survival ability in environment. Hence a study has been carried out on Isolation of fungal pathogen was done using PDA medium in order to identify the disease's causative agent and its variation in morphology and colony characters. A total of twenty-four C. gloeosporioides isolates were obtained from various Tamil Nadu mango cultiavting areas of Tamil Nadu. The isolates were identified morphologically and molecularly. The microscopic features such as conidia shape and size were used to identify C. gloeosporioides. The CTAB technique was employed to extract the DNA from the isolates of C. gloeosporioides. A total of 24 isolates were subjected to ITS (Inter Transcribed Spacer) of 18s ribosomal RNA amplification; all of these isolates exhibited amplified products with a size range of 560 bp, indicating that they were fungal pathogen. The MkCgF and MkCgR PCR primer combination was unique to the ITS region of C. gloeosporioides species. It has been confirmed that all 24 of the C. gloeosporioides isolates are really C. gloeosporioides by amplifying the predicted size of 380 bp.

Efficacy of Bacillus licheniformis: a biocontrol agent against Colletotrichum gloeosporioides Penz. (Penz. & Sacc.) causing anthracnose in greater yam (Dioscorea alata L.)

BackgroundAnthracnose, caused by Colletotrichum gloeosporioides, is the most dangerous fungal disease of greater yam (Dioscorea alata L.), and leads to significant crop loss. Microbial technology in cropping systems is currently focused on biological control agents (BCAs), which include plant endophytes like bacteria, which contribute well to sustainable production. In the present study, the ability of an endophytic bacterium, Bacillus licheniformis (CTCRI EB12), isolated from Aloe vera was investigated to prevent the disease.ResultsThe bacterium, CTCRI EB12, was characterized as a member of the Bacillus genus by morphological and biochemical tests and confirmed as B. licheniformis via 16S ribosomal gene analysis. The effectiveness of the endophytic bacterium was affirmed through cell confrontation assays and scanning electron microscopy. In cell confrontation assay, the isolate inhibited all the three C. gloeosporioides isolates used in the study with more than 80% inhibition. The effect of culture filtrate of the isolate was also tested against pathogens that were compliant with the outcome of dual culture technique. The culture and culture filtrate could effectively restrain the spreading of C. gloeosporioides in greater yam leaf. The isolate could completely inhibit the growth of the pathogen through an antibiosis study, and the same could be explained through scanning electron microscopy results, in which the endophyte-treated C. gloeosporioides hyphae appeared longer and distorted compared to control and conidia possess disfigurement in endophyte-treated sample when compared to control.ConclusionsThe study showed that Bacillus licheniformis (CTCRI EB12), the endophytic bacterium that has the capacity to enhance the protection of greater yam plants, could be further developed as a microbial pesticide to reduce the effect of anthracnose in greater yam fields. Additional investigation will reveal the endophytic isolate's antifungal capabilities and assessment of their performance in the open field. The present study depicted that it might be a potent biological control agent against C. gloeosporioides causing greater yam anthracnose.

Resistance risk and molecular mechanism associated with resistance to picoxystrobin in Colletotrichum truncatum and Colletotrichum gloeosporioides

Anthracnose, caused by Colletotrichum truncatum and C. gloeosporioides, is amongst the most serious diseases of soybean in China. Picoxystrobin, a quinone outside inhibitor fungicide, is commonly used for the control of anthracnose. Its resistance risk and mechanism in C. truncatum and C. gloeosporioides are unclear. In this study, the sensitivities of 128 C. truncatum and 121 C. gloeosporioides isolates to picoxystrobin were investigated, and unimodal distributions were observed with average EC50 values of 0.7740 and 1.1561 μg mL−1, respectively. Eleven picoxystrobin-resistant mutants of C. truncatum and six mutants of C. gloeosporioides were acquired, with EC50 values varying from 5.40–152.96 and 13.53–28.30 μg mL−1, respectively. Compared to the parental isolates, mutants showed similar or higher relative fitness in conidial production and germination, and pathogenicity. Collectively, the resistance risk of C. truncatum and C. gloeosporioides to picoxystrobin is moderate to high. There was positive cross-resistance between picoxystrobin and pyraclostrobin, but not between picoxystrobin and fluazinam, difenoconazole, or propiconazole. The G143S mutation in Cyt b protein was detected in seven high-resistant mutants of C. truncatum (RF>100), and G137R occurred in four moderate-resistant mutants (RF<50). Contrastingly, there were no point mutations in Cyt b of any C. gloeosporioides mutants. Molecular docking confirmed that two mutations conferred different resistance levels to picoxystrobin. Under greenhouse trials, picoxystrobin did not control mutants with the G143S mutation, those bearing G137R or no point mutation were somewhat controlled, but at a lower level compared to wild-type isolates. These results showed that integrated management strategies should be implemented to preserve fungicide effectiveness.

Multilocus Phylogenetic Analyses of Colletotrichum gloeosporioides Species Complex Causing Crown Rot on Strawberry in Florida.

Colletotrichum gloeosporioides is the causal agent of Colletotrichum crown rot of strawberry in the southern United States. Recent multigene studies defined C. gloeosporioides as a complex species comprised of 37 species. In our study, we phylogenetically characterized C. gloeosporioides isolates from strawberry and other noncultivated plants around strawberry fields. One hundred fifteen strawberry isolates and 38 isolates from noncultivated hosts were sequenced for five genomic regions: internal transcribed spacer, actin, calmodulin, chitin synthase, and glyceraldehyde-3-phosphate dehydrogenase. Phylogenetic analysis using the maximum likelihood and Bayesian inference methods, based on partition-specific models, revealed that most of the isolates in Florida (86%) were closely related to C. siamense, whereas 14 isolates were closely related to C. theobromicola (syn. C. fragariae), four isolates were C. fructicola, and three isolates were C. clidemiae. However, only the first three species were pathogenic to strawberry. Morphological characteristics evaluated show that mycelial growth of all species is approximately 5 mm/day, but colony morphology varies by species and incubation conditions. In vitro mating of the isolates demonstrated that C. fructicola is homothallic whereas C. siamense and C. theobromicola isolates are heterothallic. The biological importance of these different Colletotrichum species is currently being investigated to determine whether different management strategies are needed in strawberry production fields.

Rapid detection of the E198A mutation of carbendazim-resistant isolates in Colletotrichum gloeosporioides by loop-mediated isothermal amplification

Apple bitter rot is a serious agricultural disease caused by Colletotrichum gloeosporioides. In recent years, carbendazim-resistant C. gloeosporioides strains bearing an E198A point mutation in the β-tubulin gene (GAG to GCG) have emerged, threatening global apple production. As such, rapidly detecting the presence of this E198A mutation in C. gloeosporioides isolates is essential in order to monitor the spread of this pathogen and to prevent outbreaks of disease. Herein, we developed a simple loop-mediated isothermal amplification (LAMP) approach to detecting the E198A mutation in C. gloeosporioides isolates from ‘Gala’ apple samples. This optimized LAMP protocol was sufficient to establish the E198A genotype of a given isolate following a 60 min incubation at 63 °C by using four specific primers. The results of this reaction could be interpreted visually based on a fluorescent yellow-green color change upon the addition of the SYBR Green I dye, and were additionally confirmed via gel electrophoresis. Importantly, this LAMP assay was capable of rapidly and reliably detecting apples that were infected with carbendazim-resistant isolates harboring this E198A mutation. In conclusion, this LAMP assay in this study can rapidly, specifically, and sensitively detect cases of apple bitter rot caused by C. gloeosporioides isolates harboring the E198A mutation.

Occurrence and infective potential of Colletotrichum gloeosporioides isolates associated to Citrus limon var Eureka

A collection of 37 fungi associated to Italian lemon plants with disease symptoms, was obtained. Ten genera including Aspergillus, Alternaria, Nigrospora, Lasiodiplodia, Dothideomycetes, Pleurostoma, Setosphaeria, Penicillium, Fusarium and Colletotrichum were identified by using ITS1–5.8S–ITS2, D1/D2 26S and COX1 loci. The last three genera were abundant on the damaged fruits, being Colletotrichum the more abundant (32.4 %). CaInt2 and CgInt primers support the identity of these isolates as C. gloeosporioides. Variability, inferred by rep-PCR and multilocus sequence analysis shows genetic differences among the C. gloeosporioides isolates. Infective profile evaluated in Colletotrichum isolates shows different leave infection percentages (26 to 60 %). SEM analysis showed mycelium, spores and appressoria on the leaves of selected Colletotrichum isolates. Specifically, the AL-05 and AL-13 isolates showed a high chitin deacetylase activity (CDA) peaking at 1.2 U/mg protein in AL-13. This is the first report on C. gloeosporioides infecting Italian lemon leaves in Mexico.

Validation of a Decision Support System for Blueberry Anthracnose and Fungicide Sensitivity of Colletotrichum gloeosporioides Isolates.

Blueberry is an increasingly important crop in Florida. Anthracnose fruit rot (AFR), caused mostly by Colletotrichum gloeosporioides, is favored by long wetness periods and temperatures from 15 to 27°C. Currently, the model in the Strawberry Advisory System (StAS) guides fungicide applications targeting strawberry AFR. Given the similarity between blueberry and strawberry AFR, we hypothesized that the model used in StAS could be used in a decision support system (DSS) built for blueberry AFR. There is no information on inhibition posed by fungicides on C. gloeosporioides isolates from blueberry. Our objectives were to demonstrate that the model used in the StAS could be used for blueberry AFR management in Florida and to assess the sensitivity of isolates to fungicides. Four trials were undertaken in blueberry fields in Florida during two seasons to compare the effectiveness of fungicide applications according to the model with that of the growers' standard calendar. Sensitivity of blueberry C. gloeosporioides isolates to azoxystrobin, benzovindiflupyr, penthiopyrad, pydiflumetofen, boscalid, thiophanate-methyl, fluazinam, and fludioxonil was evaluated. AFR incidence and yield were compared between treatments. Following recommendations from the model resulted in disease control as effective as the standard program and in some cases with fewer applications. All isolates were sensitive to benzovindiflupyr, penthiopyrad, fluazinam, and fludioxonil. Low frequency of in vitro inhibition of isolates by azoxystrobin, pydiflumetofen, boscalid, and thiophanate-methyl should raise concern about fungicide resistance. Our results indicate that the model used in StAS could be used in a DSS to help Florida growers to manage AFR in blueberry.

Inherent tolerance of Colletotrichum gloeosporioides to fludioxonil

Colletotrichum spp. cause devastating diseases in agricultural crops, including fruit crops. They can differ in host plant and plant organ specificity and even in fungicide sensitivity. In strawberry, members of the C. gloeosporioides species complex (referred to as C. gloeosporioides) primarily cause crown rot and those of the C. acutatum species complex (referred to as C. acutatum) primarily cause fruit rot. Fludioxonil is registered for use (in combination with cyprodinil; Switch 62.5WG in the US) in strawberry against anthracnose disease caused by Colletotrichum spp. In this study we examined the sensitivity of C. gloeosporioides (C. fructicola and C. siamense) and C. acutatum (C. nymphaeae and C. fioriniae) isolates from different hosts and different geographical locations in the US to fludioxonil and examined possible mechanisms of inherent fungicide tolerance. The dose response to fludioxonil of C. gloeosporioides isolates (including 4 isolates of C. theobromicola) revealed about 70% inhibition of mycelial growth at 1 mg/L that was maintained at 10 mg/L and 100 mg/L and lead to minimum inhibitory concentration (MIC) values >100 mg/L. In contrast, mycelial growth of C. acutatum isolates was completely inhibited at 1 mg/L. C. gloeosporioides isolates were also significantly less sensitive to iprodione. An investigation into possible mechanisms of C. gloeosporioides isolates tolerance to fludioxonil and iprodione revealed no evidence of OS-1 gene involvement. Isolates of both species complexes were equally sensitive to salt stress based on mycelial growth inhibition on potato dextrose agar amended with 2%, 4%, and 6% NaCl. In addition, orthologous amino acid alterations in OS-1 previously linked to fludioxonil resistance in Botrytis cinerea were not found in C. gloeosporioides or C. acutatum isolates. This study also showed limited in vitro inhibitory activity of cyprodinil against isolates of both species complexes (MIC values >100 mg/L) and unveils a potential weakness of the fludioxonil+cyprodinil premixture marketed as Switch 62.5WG against C. gloeosporioides species complexes.

In vitro effect of four fungicides on Colletotrichum gloeosporioides causing anthracnosis on the Red Globe grape variety

Anthracnose is considered one of the most important diseases of grape crops. In this study, we evaluated the in vitro effect on potato dextrose agar (PDA) of four fungicides: benomyl (Benlate® 50 WP), carbendazim (Belico ® 500 SC), chlorothalonil (Odeon® 720 SC), and dodine (Syllit ® 400 SC) using three concentrations on a Colletotrichum gloeosporioides isolate. This species complex causes anthracnose on the Red Globe grape variety. The concentrations of each fungicide were: benomyl at 0.5, 0.25, and 1 g.L-1; carbendazim at 0.6, 0.3, and 1.2 mL.L-1; chlorothalonil at 2.4, 1.2, and 4.8 mL.L-1, and dodine at 1.6, 0.8, and 3.2 mL.L-1. Evaluated the inhibition of mycelial growth and the sporulation of the fungus. The fungus mycelium exposed to the commercial dose of the fungicides was observed using environmental scanning electron microscopy (ESEM). We used a completely randomized design with 13 treatments and five repetitions. Our results showed a degree of differential efficacy for the four fungicides and their effects were directly proportional to the dose used, benomyl and chlorothalonil being the most efficient in controlling the mycelial growth of the fungus. At the commercial dose, sporulation was inhibited at a higher rate by benomyl showing no statistically significant differences with the other products except chlorothalonil and dodine when the recommended dose was reduced by half. The damage observed by ESEM on the fungus mycelium ratified the effect obtained in vitro, i.e., deformed hyphae and irregular growth. The results of this study, complemented by electron microscopy, are promising for the appropriate selection of the best fungicides for controlling anthracnose on grape fruits, subject to the timely and correct diagnosis of the disease.

Efficacy and Baseline Sensitivity of Succinate-Dehydrogenase-Inhibitor Fungicides for Management of Colletotrichum Crown Rot of Strawberry.

The current management of Colletotrichum crown rot (CCR) of strawberry, caused by Colletotrichum gloeosporioides sensu lato, relies on the use of a few fungicide classes, particularly QoI fungicides. Since resistance to QoI fungicides has recently been detected, alternative fungicide groups are needed to control this disease. Our objective was to evaluate the efficacy of succinate-dehydrogenase-inhibitor (SDHI) fungicides in managing CCR. Five SDHI fungicides, fluopyram, isofetamid, penthiopyrad, fluxapyroxad, and benzovindiflupyr, were applied 2 days before or 1 day after inoculation of cultivar Strawberry Festival. SDHI treatments were compared with the most common fungicides used for CCR management, i.e., thiophanate-methyl, pyraclostrobin, and captan. Benzovindiflupyr applied 1 day after inoculation was effective in reducing plant mortality and disease development. The baseline sensitivity of C. gloeosporioides isolates was determined in vitro using a spiral gradient dilution assay. The EC50 for benzovindiflupyr and penthiopyrad varied from 0.08 to 1.11 and 0.45 to 3.17 µg/ml, respectively, whereas the other SDHI fungicides did not inhibit fungal growth. If registered, benzovindiflupyr could serve as an alternative to manage CCR in Florida.

Predictive Model for the Effect of Environmental Conditions on the Postharvest Development of Colletotrichum gloeosporioides Strains Isolated from Papaya (Carica papaya L.)

Colletotrichum species are the most important postharvest spoilage fungi of papaya fruit. The objective of this research was to evaluate the effect of temperature and relative humidity on growth rate and time for growth to become visible of five strains of Colletotrichum gloeosporioides isolated from papaya fruit in a complex medium. As a primary model, the radial growth rates were estimated using the Baranyi and Roberts model in papaya agar. The Solver MS Excel function was used to obtain the time to visible mycelium (tv). Secondary models obtained with the Rosso et al. cardinal model of inflection were applied to describe the effect of temperature on the growth rate (μ). The Arrhenius-Davey model was used to model tv. The obtained models seem to be satisfactory for describing both μ and tv. The relative humidity had an effect on μ and tv for all tested C. gloeosporioides isolates, but no model accurately described the behavior of the fungus. External validation of models was performed with papaya fruit. Growth models were developed with the same models used in vitro. The bias and the accuracy factors as indices for performance evaluation of predictive models in food microbiology as a function of temperature and RH were 1.22 and 1.33, respectively, for μ and 1.18 and 1.62, respectively, for tv, indicating accurate predictions. The supply chain of papaya is complex and requires constant conditions, and poor conditions can result in damage to the fruit. Knowledge of the behavior of C. gloeosporioides on papaya fruit and application of the developed models in the supply chain will help to establish transport control strategies to combat these fungi. This research has contributed to development of the first models of growth for C. gloeosporioides in Mexico.

English

Anthracnose disease is a major constraint to yam production in tropical West Africa and anywhere the crop is cultivated. This study determined the cultural characteristics and growth rates of mycelia and also characterised 6 isolates of Colletotrichum gloeosporioides, the causal agent of the yam anthracnose disease, obtained from Dioscorea rotundata leaves, vines and setts in the Tolon District of Ghana. The cultural characteristics and mycelial growth rates of the isolates were determined on Potato Dextrose Agar (PDA). The C. gloeosporioides isolates were characterised using polymerase chain reaction technique with the universal primer pairs ITS1/ITS4 and NS1/NS2, C. gloeosporioides species specific primer pairs CgInt/ITS4 and CgLac-f/CgLac-r, and C. acutatum species specific primer pairs CaGlu-f1/CaGlu-r1 and Ca-f1/Ca-r1. Based on the PCR, six isolates of C. gloeosporioides with distinct cultural characteristics were obtained. There were no significant differences (P &le; 0.05) in mycelial growth rates among the isolates. The C. gloeosporioides isolates produced characteristic band sizes on ITS1/ITS4, NS1/NS2, CgInt/ITS4 and CgLac-f/CgLac-r. None of the isolates produced a band on CaGlu-f1/CaGlu-r1 and Ca-f1/Ca-r1. The proper identification of C. gloeosporioides, the pathogen responsible for the D. rotundata anthracnose is important for the proper management of the disease. &nbsp; Key words: Anthracnose disease, cultural characteristics, DNA band, morphological characteristics, Mycelial growth rate, polymerase chain reaction (PCR).

Mutations and Overexpression of CYP51 Associated with DMI-Resistance in Colletotrichum gloeosporioides from Chili.

Chili anthracnose caused by Colletotrichum spp. is an annual production concern for growers in China. Sterol C14-demethylation inhibitors (DMIs, such as tebuconazole) have been widely used to control this disease for more than three decades. In the current study, of 48 isolates collected from commercial chili farms in Jiangsu Province of China during 2018 and 2019, 8 single-spore isolates were identified as Colletotrichum gloeosporioides and the rest were identified as C. acutatum. To determine whether the DMI resistance of isolates develops in the field, mycelial growth of the 48 isolates was measured in culture medium with and without tebuconazole. In all, 6 of the 8 C. gloeosporioides isolates were resistant to tebuconazole, but all 40 of the C. acutatum isolates were sensitive to tebuconazole. The fitness cost of resistance was low based on a comparison of fitness parameters between the sensitive and resistant isolates of C. gloeosporioides. Positive cross-resistance was observed between tebuconazole and difenconazole or propiconazole, but not prochloraz. Alignment results of the CgCYP51 amino acid sequences from the sensitive and resistant isolates indicated that mutations can be divided into three genotypes. Genotype I possessed four substitutions (V18F, L58V, S175P, and P341A) at the CgCYP51A gene but no substitutions at CgCYP51B, while genotype II had five substitutions (L58V, S175P, A340S, T379A, and N476T) at CgCYP51A, concomitant with three substitutions (D121N, T132A, and F391Y) at CgCYP51B. In addition, genotype III contained two substitutions (L58V and S175P) at CgCYP51A, concomitant with one substitution (T262A) at CgCYP51B. Molecular docking models illustrated that the affinity of tebuconazole to the binding site of the CgCYP51 protein from the resistant isolates was decreased when compared with binding site affinity of the sensitive isolates. Our findings provide not only novel insights into understanding the resistance mechanism to DMIs, but also some important references for resistance management of C. gloeosporioides on chili.

Control Effects of Several Fungicides on Jujube Anthracnose and Fungicide Resistance Monitoring

Among 6 fungicides, fluazinam, which had an excellent inhibitory effect on mycelial growth and spore germination, was also effective in controlling jujube anthracnose in the field. While EC50 values of metconazole and tebuconazole ranged from 0.2 to 0.9 μg/ml and from 0.4 to 1.1 μg/ml against the mycelial growth, respectively, each inhibitory effect on spore germination was low by 7.4% and 11.1% at 50.0 μg/ml. In the field test, they showed the control value of 80.5% and 77.0%, respectively. The protective fungicides, as mancozeb and folpet, which had a low inhibitory effect on the mycelial growth, but had a high inhibitory effect of spore germination, showed excellent disease control activities by 87.6% and 92.0% in the field. Showing a result of resistance monitoring conducted with the isolates of Colletotricum gloeosporioides, the resistance against carbendazim was already generated in the field, but it was thought that there was no resistance to pyraclostrobin, fluazinam and tebuconazole. However, if the resistance factor value of the population of C. gloeosporioides isolates to pyraclostrobin was high by 160.4, the diversity of the response to the fungicide in the population was high, so it should be taken more attention to the resistance management.

Specific and sensitive detection of the guava fruit anthracnose pathogen (Colletotrichum gloeosporioides) by loop-mediated isothermal amplification (LAMP) assay.

Anthracnose of guava, caused by the fungus Colletotrichum gloeosporioides, is a major factor limiting worldwide guava production. Timely and accurate detection of the pathogen is important in developing a disease management strategy. Herein, a loop-mediated isothermal amplification (LAMP) assay for the specific and sensitive detection of C. gloeosporioides was developed using primers targeting the β-tubulin 2 (TUB2) gene. The optimal reaction conditions were 64 °C for 60 min. The specificity of the method was tested against C. gloeosporioides isolates, Colletotrichum spp. isolates, and isolates of other genera. Positive results were obtained only in the presence of C. gloeosporioides, whereas no cross-reaction was observed for other species. The detection limit of the LAMP assay was 10 fg of genomic DNA in a 25 μL reaction. The LAMP assay successfully detected C. gloeosporioides in guava fruit collected in the field. The results indicate that the developed LAMP assay is a simple, cost-effective, rapid, highly sensitive, and specific tool for the diagnosis of guava anthracnose caused by C. gloeosporioides and could be useful for disease management.

Characterization and pathogenicity of Colletotrichum spp. causing citrus anthracnose in Tunisia.

In the winter and spring of 2014–2015, typical anthracnose symptoms were detected on different citrus varieties in Cap-Bon and Morneg-Tunis regions of Northern Tunisia. Surveys were conducted to determine the casual agents of these symptoms. A total of seven monosporic isolates were obtained from dark lesions on fruits, flowers, leaves and twigs of citrus from six orchards. One Colletotrichum karstii (from the C. boninense species complex) and six C. gloeosporioides isolates were identified through morphological analysis and sequencing of their ITS rDNA sequences. Pathogenicity tests with the seven isolates were performed on symptomless, detached citrus fruits (Valencia orange and Eureka lemon). All tested isolates caused anthracnose lesions after 1 week of incubation. Koch’s postulates were fulfilled by re-isolation of pathogens from the inoculated fruits. This report elucidates the diversity of anthracnose pathogens in Tunisia. This is the first report of C. karstii from citrus in Tunisia.

Molecular Detection of QoI Resistance in Colletotrichum gloeosporioides Causing Strawberry Anthracnose Based on Loop-Mediated Isothermal Amplification Assay.

Anthracnose is one of the most common diseases in strawberry plants. Colletotrichum gloeosporioides is the major cause of anthracnose in China, including Zhejiang Province. Early, specific, reliable, and time-saving detection is urgently needed to prevent the further spread of C. gloeosporioides, guiding farmers to utilize chemicals to control anthracnose. In this study, we showed that the high resistance to pyraclostrobin, caused by a point mutation at codon 143 (GGT→GCT) in the cytochrome b gene of C. gloeosporioides was prevalent in the strawberry growing regions, and we developed a loop-mediated isothermal amplification (LAMP) assay as a detection method. Primer sets S0 and S4 could be used to specifically detect C. gloeosporioides isolates and the G143A mutations, respectively. A detection limit of 10-2 ng (10 pg), which is at least 10-fold more sensitive than conventional polymerase chain reaction, was achieved by the LAMP assay. Here, we utilized lateral-flow devices (LFDs), nitrocellulose membranes that can absorb nucleic acids, to acquire the total genomic DNA of strawberry plants within 2 min. The LFD membranes were used as DNA templates for the LAMP assays to accurately detect strawberry plants infected with C. gloeosporioides. This diagnostic method for strawberry anthracnose was accomplished within 1 h, including the sample preparation and LAMP assays. Collectively, we developed a sensitive and practical method for monitoring C. gloeosporioides and its quinone outside inhibitor-resistant mutants. The LAMP assay for detection of C. gloeosporioides in strawberry plants has great potential for rapid strawberry anthracnose surveillance and will provide farmers with advice on preventing C gloeosporioides at the early stages of strawberry development.

Morphological Characterization of Fungal Leaf Spot Diseases of Mango ( Mangifera Indica L. ) and in Vitro Evaluation of the Effect of Antagonists on Its Mycelial Growth

The incidence of fungal leaf spot diseases on mango (Mangifera indica) in Oda Bultum University Mango farm and application of fungicide and a biological control measure was investigated. In addition microbial biocontrol and fungicide control against C. gloeosporioides developed for mangoes has been less reported in Ethiopia. Common mango disease control mechanisms in Ethiopia are utilization of pesticides. However, pesticides are overused and misused. As a result there is an urgent need to reduce the use of synthetic chemicals. Biological control offers an alternative to the use of pesticides. Invitro evaluation and testing of T. harzianum showed maximum mycelial growth inhibition (74.4% and 73.5%) on C. gloeosporioides isolates 1 and 3 respectively. The minimum percent of mycelial growth inhibition (71.5%) of T. harzianum was observed on isolate 1. Pseudomonas fluorescens showed maximum mycelial growth inhibition on isolate 2 (74.1%), followed by isolate 1 (70.4%). Least percent of mycelial growth inhibition by P. fluorescens was observed on isolate 3 (69%). B. subtilis showed maximum mycelial growth inhibition on isolate 3 (56.1%), followed by isolate 1 (51.5%). Least percent of mycelial growth inhibition by B. subtilis was observed on isolate 2 (48.6%). In vitro evaluation of fungicides against the three isolated test pathogen revealed that at 1000 PPM, all fungicides showed highest percentage inhibition (72.8% to 90.14%). The highest percentage inhibition was observed at the concentration of1000 PPM on isolates 2 (90.14%) and 3 (88.13%) and the lowest percentage inhibition was observed at the concentration of 200 PPM on isolates 1 (71.46%) and 3 (77.18%). The highest percentage mycelial growth inhibition by Sancozeb 80% WP on the isolates was 90.14% at a concentration of 1000PPM, whereas the lowest percentage mycelia growth inhibition of 83.06% was recorded at 200PPM. The highest percentage mycelial growth inhibition by Ridomil on the isolates was (83.16%) at 1000 PPM and the lowest percentage mycelial growth inhibition was recorded on isolate 1 (72.83%) at 200 PPM. Among the two fungicides Sancozeb was the most effective fungicide to inhibit the growth of the isolates with 87.98-90.14% inhibition.