Marine yeasts and bacteria as biological control agents against anthracnose on mango

The potential of four yeasts (Debaryomyces hansenii, Rhodotorula minuta, Cryptococcus laurentii and Cryptococcus diffluens) and three bacteria (Bacillus amyloliquefaciens, Bacillus subtilis and Stenotrophomonas rhizophila) with antagonistic capacity against anthracnose caused by Colletotrichum gloeosporioides in mango cv. Ataulfo fruit was investigated. Germination of C. gloeosporioides spores was significantly inhibited by all marine yeasts and bacteria strains of an in vitro test. When yeasts and bacteria were tested on mango fruit, the marine yeast D. hansenii 1R11CB strain and marine bacteria S. rhizophilaKM02 strain were the best antagonists to anthracnose (C. gloeosporioides), which significantly decreased disease incidence by 56% and 89%, respectively, and reduced lesion diameter by 91% and 92%, respectively. All the isolated strains of the phytopathogen, yeasts and bacteria were molecularly identified. Our results from in vitro and in vivo experiments suggest that marine yeasts and bacteria strains can be used as some effective biological control agents for anthracnose in mango.     

First report of anthracnose on noni caused by Colletotrichum gloeosporioides in India

Noni, an important medicinal plant grown in southern India suffered heavy loss due to anthracnose disease in 2008–2009. Based on their pathogenicity, morphological and cultural characters and ribosomal DNA spacer sequences, the pathogen was identified as Colletotrichum gloeosporioides. This is the first report of anthracnose on noni in India.     

The antagonistic effect and mechanisms of Bacillus amyloliquefaciens DGA14 against anthracnose in mango cv. ‘Carabao’

Bacillus amyloliquefaciens strain DGA14 was tested for in vitro antagonism towards Colletotrichum gloeosporioides, a causal pathogen of anthracnose in mango cv. ‘Carabao’. DGA14 produced extracellular metabolites in solid and liquid media that suppressed the growth of C. gloeosporioides. The cells of DGA14 were often observed adjacent to the pathogen so affecting its spore germination and mycelium development. DGA14 colonised mango fruit 48 h after artificial inoculation and persisted 14 days after storage at 18–20°C. On fruit surfaces, DGA14 attached and produced dents to spores of C. gloeosporioides. Dipping mangoes in aqueous cell suspension (10⁸ mL L^?1) of DGA14 significantly decreased the incidence of anthracnose as compared to untreated fruit.     

First report of anthracnose caused by Colletotrichum gloeosporioides on Boehravia diffusa in India

Leaf spot symptoms were observed on the leaves of Boehravia diffusa L. plants from different regions of Bilaspur, Himachal Pradesh, India. The symptoms initially appeared as a small light-pale coloured, circular spots enlarge gradually and ultimately leading to drying of the leaves. The disease was found to be caused by a fungus. The fungus was cultured on potato dextrose agar medium and identified as Colletotrichum gloeosporioides. According to the literature, this is thefirst report of anthracnose disease of B. diffusa caused by C. gloeosporioides in India.     

Rapid assessment of resistance of tissue-cultured water yam (Dioscorea alata) and white guinea yam (Dioscorea rotundata) to anthracnose (Colletotrichum gloeosporioides Penz.)

Yam anthracnose is caused by the pathogen Colletotrichum gloeosporioides Penz. and has been identified as the most important biotic constraint to yam production worldwide. Rapid assessment of the disease is vital to its effective diagnosis and management. In this study, tissue-cultured yam plantlets of five lines of Dioscorea alata and nine of D. rotundata were rapidly assessed for their reactions to two isolates of yam anthracnose. The plantlets, obtained from meristem of the nodal cuttings, were grown for 8?weeks on Murashige and Skoog (MS) basal medium, acclimatised for 3?weeks, hardened for an additional 3?weeks, arranged in screen house in completely randomised design and sprayed with spore inocula prepared from 7?day-old culture of the two strains of Colletotrichum gloeosporioidies Penz. The relative resistance of the different Dioscorea spp. was evaluated using three disease indices – severity at seventh day after inoculation, SD7; area under disease progress curve, AUDPC; and disease severity rate, Rd. A modified rank-sum classification method put TDa 1425 and TDr 2040, with rank sum of 2.0 each, as resistant. TDr 2121, TDr 2287 and TDr 2048 were susceptible with rank sum of 27.50, 25.50 and 24.50, respectively. Dioscorea alata TDa 1425 and Dioscorea rotundata TDr 2040 were recommended in areas endemic with yam anthracnose, and also as parent lines while breeding for resistance to anthracnose.     

The antagonistic action of Trichoderma harzianum strain DGA01 against anthracnose-causing pathogen in mango cv. ‘Carabao’

The mechanisms of control and efficacy of Trichoderma harzianum strain DGA01 against anthracnose-causing pathogen Colletotrichum gloeosporioides in mango cv. ‘Carabao’ were examined. The action of DGA01 towards C. gloeosporioides was mycoparasitism and production of metabolites. DGA01 parasitised the pathogen by coiling its mycelia and spores on both artificial media and mango fruit surfaces. DGA01 was a parasitic necrotroph capable of killing C. gloeosporioides in 14 days of coexistence in artificial media. Dipping fruit in conidial suspension (10⁶?mL?L^?1) of DGA01 significantly decreased the incidence of anthracnose as compared to untreated fruit. Reduction in anthracnose severity was 87.90% showing high antagonistic potential of DGA01 in vivo.     

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.     

Assessment of potential antagonists for anthracnose (Colletotrichum gloeosporioides) disease of mango (Mangifera indica L.) in North Western Ethiopia (Pawe)

Mango (Mangifera indica L.) is considered as one of the most popular fruits among millions of people in the tropical area and increasingly in the developed countries. Anthracnose, caused by the fungus Colletotrichum gloeosporioides, is the most important pre- and post-harvest disease of mango. The objective of this research was to evaluate the prevalence of different promising antagonistic Trichoderma and Bacillus spp. on phyloplane of mango in Ethiopia and to evaluate their antagonistic potential against the pathogen. A total of 19 mango fields were surveyed and anthracnose affected all fields. Culture studies on potato dextrose agar for evaluation of antibiosis activity of Trichoderma spp. and Bacillus spp. revealed that they have inhibitory and lytic effect on C. gloeosporioides, which is an indication of their potential biocontrol agent for management of mango anthracnose as an alternative to chemical control. Significant differences (p?<?0.05) were observed among Bacillus isolates in causing lysis of pathogen mycelium, when inoculated on actively growing colony of C. gloeosporioides. Maximum reduction in growth rate of pathogen was observed with Bacillus spp. (B50), which restricted the growth to 2.7?mm compared to 8.3?mm in the control with 67.5% efficacies. There were similar effects (p?<?0.05) among Trichoderma spp. in formation of inhibition zones and lysis by varying degrees up to 59.7% efficacies in reducing linear growth of the pathogen in dual culture.     

PCR-based detection and characterization of the fungal pathogens Colletotrichum gloeosporioides and Colletotrichum capsici causing anthracnose in papaya (Carica papaya l.) in the Yucatan peninsula

Colletotrichum gloeosporioides is the common causal agent of anthracnose in papaya (Carica papaya L.) fruits, and infection by this fungal pathogen results in severe post-harvest losses. In the Yucatán peninsula (Mexico) a different Colletotrichum species was isolated from papaya fruits with atypical anthracnose lesions. The DNAs from a variety of Colletotrichum isolates producing typical and atypical lesions, respectively, were amplified by PCR with C.gloeosporioides-specific primers. All isolates from typical anthracnose lesions yielded a 450 bp PCR product, but DNAs from isolates with atypical lesions failed to produce an amplification product. For further characterization, the rDNA 5.8S-ITS region was amplified by PCR and processed for sequencing and RFLP analysis, respectively, to verify the identity of the papaya anthracnose pathogens. The results revealed unequivocally the existence of two Colletotrichum species causing anthracnose lesions on papaya fruits: C. gloeosporioides and C. capsici. PCR-RFLP using the restriction endonuclease MspI reliably reproduced restriction patterns specific for C. capsici or C. gloeosporioides. The generation of RFLP patterns by MspI (or AluI or RsaI) is a rapid, accurate, and unequivocal method for the detection and differentiation of these two Colletotrichum species.     

Colletotrichum fructicola and C. siamense are involved in chilli anthracnose in India

Chilli anthracnose is a major problem in India and worldwide. In this study, we investigated the phylogenetic relationships of 52 fungal isolates associated with chilli anthracnose in southern India. All the 52 isolates were sequenced for partial ITS/5.8S rRNA and glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes and showed affinities with Colletotrichum siamense and C. fructicola within Colletotrichum. gloeosporioides species complex. Further, a reduced subset of 17 selected isolates was made and in a maximum parsimony analysis of a multigene data-set including partial ITS/5.8S rRNA, actin (act), calmodulin (cal), chitin synthase (chs1), gapdh and β-tubulin (tub2) gene sequence data, these fungal isolates clustered with the type strain of C. fructicola, except for strain MTCC 3439 that showed phylogenetic affinities with C. siamense. The pathogenicity tests involving two representative isolates: UASB-Cg-14 and MTCC 3439, confirmed the involvement of C. fructicola and C. siamense in the development of disease symptoms on fresh chilli fruits. This is the first report of the association of C. fructicola and C. siamense in causing chilli anthracnose in India.     

Germination and conjugation of Bacillus thuringiensis subsp. israelensis in the intestine of gnotobiotic rats

Aims: To study the ability of Bacillus thuringiensis subsp. israelensis spores to germinate and subsequently transfer a conjugative plasmid in the intestinal tract of gnotobiotic rats. Methods and Results: Germination was studied by feeding germ-free rats with spores of a B. thuringiensis strain harbouring a plasmid encoding green fluorescent protein (GFP), which enabled quantification of germinated bacteria by flow cytometry. To study in vivo conjugation, germ-free rats were first associated with a B. thuringiensis recipient strain and after 1 week an isogenic donor strain harbouring the conjugative plasmid pXO16 was introduced. Both strains were given as spores and transfer of pXO16 was observed from the donor to the recipient strain. Conclusions: Bacillus thuringiensis is able to have a full life cycle in the intestine of gnotobiotic rats including germination of spores, several cycles of growth and sporulation of vegetative cells. For the first time conjugative plasmid transfer in a mammalian intestinal tract was shown between two B. thuringiensis strains. Significance and Impact of the Study: Strains of B. thuringiensis are used worldwide to combat insect pests, and this study brings new insights into the nature of B. thuringiensis showing the potential of the bacteria to germinate and transfer DNA in the mammalian intestinal tract.     

Pathogenic and genetic variability among Colletotrichum gloeosporioides isolates from different yam hosts in the agroecological zones in Nigeria

Anthracnose, caused by Colletotrichum gloeosporioides Penz., is the most severe foliar disease of water yam (Dioscorea alata) worldwide. Population genetic analyses can yield useful insights into the evolutionary potential of C. gloeosporioides and thus lead to the development of appropriate disease management strategies. The genetic structure of C. gloeosporioides populations from yam and non‐yam hosts in three agroecological zones of Nigeria was investigated. Microsatellite‐primed polymerase chain reaction (MP‐PCR), virulence phenotyping using five putative D. alata differentials, cross‐inoculation tests, and the presence/absence of a Glomerella teleomorph in yam fields were used to infer the evolutionary potential of C. gloeosporioides on yam. We observed high genotypic diversity (GD = 0.99 to 1.00) for populations from all hosts and agroecological zones, with multiple pathogen genotypes in individual anthracnose lesions. Genetic differentiation was low among pathogen populations from different hosts (G_ST = 0.10, θ = 0.034), and agroecological zones (G_ST = 0.04, θ = 0.018), indicating limited host differentiation and significant gene flow. No evidence was found for the existence of C. gloeosporioides f. sp. alatae reported in previous studies. The fungus was recovered from several non‐yam host species commonly found in yam fields but non‐yam isolates caused only mild to moderate symptoms on yam. Eighteen C. gloeosporioides virulence phenotypes were identified among 217 isolates but there was a weak correlation (r = 0.02, P = 0.40) between virulence phenotype and MP‐PCR haplotype. Consistent with the above findings, we observed for the first time the Glomerella teleomorph on anthracnose‐infected yam plants in Nigeria, indicating that sexual recombination might play an important role in anthracnose epidemics on yam. The implications of these findings for C. gloeosporioides evolutionary potential and anthracnose resistance breeding are discussed.     

Mixtures of plant growth-promoting rhizobacteria for induction of systemic resistance against multiple plant diseases

Studies of induced systemic resistance using strains of plant growth-promoting rhizobacteria (PGPR) have concentrated on the use of individual PGPR as inducers against multiple diseases of a single crop. To date, few reports have examined the potential of PGPR strain mixtures to induce systemic resistance against diseases of several different plant hosts. The objective of this study was to select mixtures of compatible PGPR strains with the capacity to elicit induced systemic resistance in four hosts. The specific diseases and hosts tested in this study included: bacterial wilt of tomato (Lycopersicon esculentum) caused by Ralstonia solanacearum, anthracnose of long cayenne pepper (Capsicum annuum var. acuminatum) caused by Colletotrichum gloeosporioides, damping off of green kuang futsoi (Brassica chinensis var. parachinensis) caused by Rhizoctonia solani, and cucumber mosaic virus (CMV) on cucumber (Cucumis sativus). To examine compatibility, seven selected PGPR strains were individually tested for in vitro antibiosis against all other PGPR strains and against three of the tested pathogens (R. solanacearum, C. gloeosporioides, and R. solani). No in vitro antibiosis was observed among PGPR strains or against pathogens. Twenty-one combinations of PGPR and seven individual PGPR were tested in the greenhouse for induced resistance activity. Results indicated that four mixtures of PGPR and one individual strain treatment significantly reduced the severity of all four diseases compared to the nonbacterized control: 11 mixtures reduced CMV of cucumber, 16 mixtures reduced bacterial wilt of tomato, 18 mixtures reduced anthracnose of long cayenne pepper, and 7 mixtures reduced damping off of green kuang futsoi. Most mixtures of PGPR provided a greater disease suppression than individual PGPR strains. These results suggest that mixtures of PGPR can elicit induced systemic resistance to fungal, bacterial, and viral diseases in the four hosts tested.     

Biological control of anthracnose (Colletotrichum gloeosporioides) in yam by Streptomyces sp.MJM5763

Aim: To find a suitable biocontrol agent for yam anthracnose caused by Colletotrichum gloeosporioides. Methods and Results: An actinobacterial strain, MJM5763, showing strong antifungal activity, multiple biocontrol and plant growth‐promoting traits was isolated from a yam cultivation field in Yeoju, South Korea. Based on morphological and physiological characteristics and analysis of the 16S rDNA sequence, strain MJM5763 was identified as a novel strain of Streptomyces and was designated as Streptomyces sp. MJM5763. Treatment with MJM5763 and the crude culture filtrate extract (CCFE) was effective in suppressing anthracnose in detached yam leaves in vitro and reduced incidence and severity of anthracnose in yam plants under greenhouse conditions. The CCFE treatment was the most effective of all the treatments and reduced the anthracnose severity by 85–88% and the incidence by 79–81%, 90 days after inoculation with the pathogen. CCFE treatment was also effective under field conditions and showed a reduction of 86 and 75% of anthracnose severity and incidence, respectively. Conclusion: Streptomyces sp. strain MJM5763 was effective in biocontrolling anthracnose in yam caused by C. gloeosporioides. Significance and Impact of the Study: Streptomyces sp. MJM5763 is a potential alternative to chemical fungicides for reducing yield losses to anthracnose in yam.     

Genetic variability in mulberry against Myrothecium leaf spot (Myrothecium roridum) and identification of resistance genotypes

Disease response of 30 mulberry genotypes to Myrothecium leaf spot (Myrothecium roridum) was studied under inoculated condition. It was observed that 10 genotypes were resistant, 16 genotypes moderately resistant and 4 genotypes moderately susceptible to the disease. Area under disease progress curve (AUDPC) and apparent infection rate was found significantly lower in the resistant genotypes. Correlation study revealed that percent disease index (PDI) has significant positive correlation with AUDPC and apparent infection rate. Genetic analysis of disease-resistant traits (PDI and mean AUDPC) revealed that phenotypic coefficient of variation (PCV) was higher than genotypic coefficient of variation (GCV) for both PDI and AUDPC, and GCV/PCV ratio was also found high which indicated that disease-resistant traits to Myrothecium leaf spot were not much influenced by environment. High heritability coupled with high genetic advance indicated that the disease-resistant traits are due to additive gene effect thereby indicating the amenability of disease-resistant in the selection process. Hence disease-resistant mulberry genotypes viz. C-763, S-34, Jodhpur, Cyprus, Australian and Hungarian may be used as source of resistance to Myrothecium leaf spot for future breeding programme. Besides, high yielding genotypes viz. Tr-10, C-763 and S-34 may be recommended for commercial exploitation     

Antifungal effects of dimethyl trisulfide against Colletotrichum gloeosporioides infection on mango

Colletotrichum gloeosporioides, one of the main agents of mango anthracnose, causes latent infections in unripe mango and can lead to huge losses during fruit storage and transport. Dimethyl trisulfide (DMTS) is an antifungal agent produced by several microorganisms or plants, but its effects on the infection process of C. gloeosporioides have not been well characterized. A histological investigation demonstrated that DMTS exhibits strong inhibitory effects on the infection process of C. gloeosporioides in planta by inhibiting the germination of conidia and formation of appressoria, damaging cytoplasm to cause cells to vacuolate and contributing to deformation of appressoria prior to penetration. This is the first study to demonstrate antifungal activity of DMTS against C. gloeosporioides on mango by suppression of the infection process, thus providing a novel postharvest biorational control for mango anthracnose.     

Two Genetically Distinct Populations of Colletotrichum gloeosporioides Penz. Causing Anthracnose Disease of Yam (Dioscorea spp.)

Variation within Colletotrichum gloeosporioides, the causal agent of yam anthracnose disease, is still poorly defined and this hinders breeding for resistance. Two morphotypes of C. gloeosporioides, designated slow‐growing grey (SGG) and fast‐growing salmon (FGS), are associated with anthracnose disease of yam in Nigeria. The morphotypes are distinguishable based on colony and conidial morphology, growth rate, virulence, as well as vegetative compatibility, but molecular differentiation of SGG and FGS strains is needed to facilitate epidemiological studies. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)‐amplified small subunit (18S) rDNA fragments, and microsatellite‐primed PCR (MP‐PCR) genomic fingerprinting were employed to provide a basis for molecular differentiation of the morphotypes. DGGE analysis revealed patterns that clearly differentiated isolates of the aggressive defoliating SGG from the moderately virulent non‐defoliating FGS strains. Genetic analysis based on 52 MP‐PCR markers revealed highly significant differentiation between the SGG and FGS populations on yam (G_ST = 0.22; Nei's genetic identity = 0.85; θ = 0.28, P < 0.001), indicating that the SGG and FGS morphotypes represent genetically differentiated populations. The results of the molecular typing using DGGE and MP‐PCR analyses were consistent with the disease phenotype caused by the two morphotypes. Consequently, these molecular techniques might be used, at least partly, to replace time‐consuming virulence studies on yam.     

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

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