Colletotrichum Lagenarium Research Articles

Design, Synthesis and Antifungal Activities of Novel Strobilurin Derivatives Containing Pyrimidine Moieties

Strobilurins are one of the most important classes of agricultural fungicides. To discover new strobilurin derivatives with high activity against resistant pathogens, a series of novel beta-methoxyacrylate analogues were designed and synthesized by integrating substituted pyrimidine with a strobilurin pharmacophore. The compounds were confirmed and characterized by infrared, H-1 nuclear magnetic resonance, elemental analysis and mass spectroscopy. The bioassays indicated that most of the compounds (1a-1h) exhibited potent antifungal activities against Colletotrichum orbiculare, Botrytis cinerea Pers and Phytophthora capsici Leonian at the concentration of 50 mu g/mL. Exhilaratingly, compound 1d (R=3-trifluoromethylphenyl) showed better antifungal activity against all the tested fungi than the commercial strobilurin fungicide azoxystrobin.

Sesquiterpenes from the marine red alga Laurencia composita

Four new chamigrane derivatives, laurecomin A (1), laurecomin B (2), laurecomin C (3), and laurecomin D (4), one new naturally occurring sesquiterpene, 2,10-dibromo-3-chloro-7-chamigren-9-ol acetate (5), and three known halogenated structures, deoxyprepacifenol (6), 1-bromoselin-4(14),11-diene (7), and 9-bromoselin-4(14),11-diene (8), were isolated from the marine red alga Laurencia composita collected from Pingtan Island, China. The structures of these compounds were unambiguously established by 1D, 2D NMR and mass spectroscopic techniques. The bioassay results showed that 2 was active against both brine shrimp and fungus Colletotrichum lagenarium.

Synthesis and Antifungal Activities of New Type β‐Methoxyacrylate‐Based Strobilurin Analogues

AbstractStrobilurins have become one of the most important classes of agricultural fungicides. To discover new strobilurin derivatives with high activity against resistant pathogens, a series of novel β‐methoxyacrylate analogues were designed and synthesized by integrating substituted pyrimidine with a strobilurin pharmacophore. The compounds were confirmed and characterized by infrared, 1H nuclear magnetic resonance, elemental analysis and mass spectroscopy. The bioassays indicated that most of the compounds 1 exhibited potent antifungal activity against Colletotrichum orbiculare, Botrytis cinerea Pers and Phytophthora capsici Leonian at the concentration of 50 μg/mL. Exhilaratingly, compound 1a (R=methyl) showed better antifungal activity against all the tested fungi than the commercial strobilurin fungicide azoxystrobin.

Characterization of a fungal thioesterase having claisen cyclase and deacetylase activities in melanin biosynthesis

Cell wall melanins are darkly-pigmented macromolecules that contribute to pathogenicity. In fungi, melanin biosynthesis generally involves polymerization of 1,8-dihydroxynaphthalene via a 1,3,6,8-tetrahydroxynaphthalene (THN) precursor whose backbone is assembled by iterative, nonreducing polyketide synthases. Convergent routes to THN have evolved in fungi. Parallel heptaketide and hexaketide pathways utilize conventional C-terminal thioesterase/Claisen cyclase domains and discrete side-chain deacylases. In contrast, in vitro characterization of Pks1 from Colletotrichum lagenarium establishes a true THN synthase with a bifunctional thioesterase (TE) catalyzing both cyclization and deacetylation of an enzyme-bound hexaketide. Chimeric TE domains were generated by swapping lid regions of active sites between classes of melanin-related TEs to gain insight into this unprecedented catalysis of carbon-carbon bond making and breaking by a single α/β-hydrolase fold enzyme.

Evaluation of antifungal activity of seaweed extracts

Seaweeds are subject to numerous biological interactions and sometimes to extreme abiotic conditions, so they have developed among other defense mechanisms, the ability to produce biologically active substances. Thus, these organisms produce mainly terpenes and phenols. Among others, the antifungal activity, due to its importance in human and animal health and the production of agricultural products, has been the subject of several studies. In the present work, this activity was investigated in ten seaweeds extracts, by direct bioautography assays, compared to Colletotrichum lagenarium and disk diffusion assay, compared to Aspergillus flavus. The organisms studied were: Stypopodium zonale, Laurencia dendroidea, Ascophyllum nodosum, Sargassum muticum, Pelvetia canaliculata, Fucus spiralis, Sargassum filipendula, Sargassum stenophyllum, Laminaria hyperborea and Gracilaria edulis. S. zonale, L. dendroidea, P. canaliculata, S. muticum, A. nodosum and F. spiralis extracts significantly inhibited the C. lagenarium growth, but not inhibited significantly the A. flavus growth. The presence of terpenes in all of these extracts was confirmed by thin layer chromatography whereas the presence of phenolic compounds was confirmed only in extracts of P. canaliculata, A. nodosum and S. muticum. In chemical study by column chromatography, followed by gas chromatography/mass spectrometry analysis, the terpenes neophytadiene, cartilagineol, obtusol elatol; and the ester ethyl hexadecanoate were identified in the L. dendroidea extract. This is the first report on the activity of seaweed extracts against C. lagenarium, a fungus bearing agricultural importance.

Genomic sequence-based discovery of novel angucyclinone antibiotics from marine Streptomyces sp. W007

A large number of novel bioactive compounds were discovered from microbial secondary metabolites based on the traditional bioactivity screenings. Recent fermentation studies indicated that the crude extract of marine Streptomyces sp. W007 possessed great potential in agricultural fungal disease control against Phomopsis asparagi, Polystigma deformans, Cladosporium cucumerinum, Monilinia fructicola, and Colletotrichum lagenarium. To further evaluate the biosynthetic potential of secondary metabolites, we sequenced the genome of Streptomyces sp. W007 and analyzed the identifiable secondary metabolite gene clusters. Moreover, one gene cluster with type II PKS implied the possibility of Streptomyces sp. W007 to produce aromatic polyketide of angucyclinone antibiotics. Therefore, two novel compounds, 3-hydroxy-1-keto-3-methyl-8-methoxy-1,2,3,4-tetrahydro-benz[α]anthracene and kiamycin with potent cytotoxicities against human cancer cell lines, were isolated from the culture broth of Streptomyces sp. W007. In addition, other four known angucyclinone antibiotics were obtained. The gene cluster for these angucyclinone antibiotics could be assigned to 20 genes. This work provides powerful evidence for the interplay between genomic analysis and traditional natural product isolation research.

Large-Scale Gene Disruption in Magnaporthe oryzae Identifies MC69, a Secreted Protein Required for Infection by Monocot and Dicot Fungal Pathogens

To search for virulence effector genes of the rice blast fungus, Magnaporthe oryzae, we carried out a large-scale targeted disruption of genes for 78 putative secreted proteins that are expressed during the early stages of infection of M. oryzae. Disruption of the majority of genes did not affect growth, conidiation, or pathogenicity of M. oryzae. One exception was the gene MC69. The mc69 mutant showed a severe reduction in blast symptoms on rice and barley, indicating the importance of MC69 for pathogenicity of M. oryzae. The mc69 mutant did not exhibit changes in saprophytic growth and conidiation. Microscopic analysis of infection behavior in the mc69 mutant revealed that MC69 is dispensable for appressorium formation. However, mc69 mutant failed to develop invasive hyphae after appressorium formation in rice leaf sheath, indicating a critical role of MC69 in interaction with host plants. MC69 encodes a hypothetical 54 amino acids protein with a signal peptide. Live-cell imaging suggested that fluorescently labeled MC69 was not translocated into rice cytoplasm. Site-directed mutagenesis of two conserved cysteine residues (Cys36 and Cys46) in the mature MC69 impaired function of MC69 without affecting its secretion, suggesting the importance of the disulfide bond in MC69 pathogenicity function. Furthermore, deletion of the MC69 orthologous gene reduced pathogenicity of the cucumber anthracnose fungus Colletotrichum orbiculare on both cucumber and Nicotiana benthamiana leaves. We conclude that MC69 is a secreted pathogenicity protein commonly required for infection of two different plant pathogenic fungi, M. oryzae and C. orbiculare pathogenic on monocot and dicot plants, respectively.

Cell Death ofNicotiana benthamianaIs Induced by Secreted Protein NIS1 ofColletotrichum orbiculareand Is Suppressed by a Homologue of CgDN3

Colletotrichum orbiculare, the causal agent of cucumber anthracnose, infects Nicotiana benthamiana. Functional screening of C. orbiculare cDNAs in a virus vector-based plant expression system identified a novel secreted protein gene, NIS1, whose product induces cell death in N. benthamiana. Putative homologues of NIS1 are present in selected members of fungi belonging to class Sordariomycetes, Dothideomycetes, or Orbiliomycetes. Green fluorescent protein-based expression studies suggested that NIS1 is preferentially expressed in biotrophic invasive hyphae. NIS1 lacking signal peptide did not induce NIS1-triggered cell death (NCD), suggesting apoplastic recognition of NIS1. NCD was prevented by virus-induced gene silencing of SGT1 and HSP90, indicating the dependency of NCD on SGT1 and HSP90. Deletion of NIS1 had little effect on the virulence of C. orbiculare against N. benthamiana, suggesting possible suppression of NCD by C. orbiculare at the postinvasive stage. The CgDN3 gene of C. gloeosporioides was previously identified as a secreted protein gene involved in suppression of hypersensitive-like response in Stylosanthes guianensis. Notably, we found that NCD was suppressed by the expression of a CgDN3 homologue of C. orbiculare. Our findings indicate that C. orbiculare expresses NIS1 at the postinvasive stage and suggest that NCD could be repressed via other effectors, including the CgDN3 homologue.

Synthesis and Antifungal Activities of Carabrol Ester Derivatives

Thirty-eight new ester derivatives of carabrol were designed, synthesized, and characterized by (1)H and (13)C NMR and HR-ESI-MS. Their antifungal activities against the fungal pathogen Colletotrichum lagenarium were evaluated using a spore germination assay. Of these 38 ester derivatives, 16 showed higher antifungal activity than that of carabrol and 7 showed higher antifungal activity than that of carabrone. It was found that the C-4 position of carabrol was a key position involving its antifungal activity, which showed the variation of 50% inhibition concentration (IC(50)) from 2.70 to 52.33 μg/mL. When substituted by the phenyl ring, the ester derivatives with electron-attracting groups showed higher activity than those with electron-donating ones. Two ester derivatives, carabryl 4-cynaobenzoate (II-17, IC(50) 2.70 μg/mL) and carabryl 4-isopropylbenzoate (II-27, IC(50) 2.82 μg/mL), showed only slightly lower antifungal activity than that of the positive control chlorothalonil (IC(50) 0.87 μg/mL) and have been identified as promising leads for development of new environmentally friendly fungicides.

Anthracnose and Gummy Stem Blight Are Reduced on Watermelon Grown on a No-Till Hairy Vetch Cover Crop.

Multiple applications of fungicides are used to manage anthracnose caused by Colletotrichum orbiculare and gummy stem blight caused by Didymella bryoniae, the two most common and destructive diseases on watermelon (Citrullus lanatus) in the mid-Atlantic region of the United States. To develop a sustainable, nonchemical management option, a split-plot experiment was conducted over 3 years to evaluate the effects of a no-till hairy vetch (Vicia villosa) cover crop on disease severity, plant growth, and fruit yield compared with two conventional bedding systems and fungicide application. The main plots were bedding strategies consisting of bare ground, polyethylene covering, or a hairy vetch cover crop that was planted in the fall, killed the following spring, and left on the soil surface as an organic mulch. The subplots were a nonfungicide control or a weekly application of a standard fungicide program. Hairy vetch mulch provided greater than a 65% reduction in the area under the disease progress curves of anthracnose and gummy stem blight and greater than an 88% decrease in diseased fruit compared with bare ground or polyethylene mulch. The reductions were comparable with those achieved by fungicide applications. Watermelon vine lengths in plots with hairy vetch were similar to or greater than those in plots with polyethylene or bare ground that were treated with fungicides. Marketable fruit in plots with hairy vetch was higher compared with bare ground in 2 of 3 years and was similar to that in plots treated with fungicides in all 3 years. Addition of fungicide application to hairy vetch treatment further reduced anthracnose in 1 year and gummy stem blight in 2 years but did not significantly increase fruit yield in all 3 years. This is the first demonstration that a no-till hairy vetch production system can reduce anthracnose and gummy stem blight on watermelon and that the production system has the potential to mitigate damage caused by these diseases.

Synthesis, Structure and Antifungal Activity of New 3-[(5-Aryl-1,3,4-oxadiazol-2-yl)methyl]benzo[d]thiazol-2(3H)-ones

A series of new 3-[(5-aryl-1,3,4-oxadiazol-2-yl)methy])benzo[d]thiazol-2(3H)-ones were synthesized by reaction of (5-substituted-2-oxobenzothiazolin-3-yl)-acetohydrazide with various aromatic acids in POCl3 under reflux conditions. The structures of the title compounds were confirmed by 1H-NMR, 13C-NMR, IR, MS and elemental analysis. Furthermore, the structure of compound 4i was determined by single-crystal X-ray diffraction. The preliminary bioassy results indicated that some of them showed moderate inhibition activity against Colletotrichum orbiculare, Botrytis cinerea and Rhizoctonia solani.

Antifungal activity of plant extracts against Colletotrichum lagenarium, the causal agent of anthracnose in cucumber

Colletotrichum lagenarium is an important plant-pathogenic fungus that causes anthracnose of cucumber, a disease that is widespread under both greenhouse and field cultivation. To find a promising method for the control of this disease, extracts of eight plants from China were screened in the study presented here. The results showed that the extract of Cinnamomum camphora (L.) Presl had significantly greater inhibitory activity against C. lagenarium than the other seven plant extracts tested in vitro. At 5 days after inoculation in a potted seedling experiment, C. camphora extract demonstrated 95% control of C. lagenarium at a concentration of 16 mg mL⁻¹, and the MIC₅₀ was 2.596 mg mL⁻¹. Microscopic observation showed that this extract had a significant impact on the morphology of the fungus, leading to shorter hyphae that were more branched; this observation was associated with the inhibition of mycelial growth. Stability assays revealed that the extract was relatively stable at 80 °C, under acidic conditions and when exposed to light and short periods of UV radiation. These results demonstrated that the extract from C. camphora could be used as a potent phytochemical fungicide.

Isolation of Streptomyces Strain STS1 that Inhibits the Growth of Colletotrichum orbiculare

Isolation of Streptomyces Strain STS1 that Inhibits the Growth of Colletotrichum orbiculare

Novel Benzo-1,2,3-thiadiazole-7-carboxylate Derivatives As Plant Activators and the Development of Their Agricultural Applications

Plant activators are a novel kind of agrochemicals that could induce resistance in many plants against a broad spectrum of diseases. To date, only few plant activators have been commercialized. In order to develop novel plant activators, a series of benzo-1,2,3-thiadiazole-7-carboxylate derivatives were synthesized, and the structures were characterized by (1)H NMR, IR, elemental analyses, and HRMS or MS. Their potential systemic acquired resistance as plant activators was evaluated as well. Most of them showed good activity, especially, fluoro-containing compounds 3d and 3e, which displayed excellent SAR-inducing activity against cucumber Erysiphe cichoracearum and Colletotrichum lagenarium in assay screening. Field test results illustrated that compounds 3d and 3e were more potent than the commercial plant activator, S-methyl benzo[1,2,3]thiadiazole-7-carbothioate (BTH) toward these pathogens. Further, the preparation of compound 3d is more facile than BTH with lower cost, which will be helpful for further applications in agricultural plant protection.

Promoção de crescimento e indução de resistência à antracnose por Trichoderma spp. em pepineiro

O objetivo deste trabalho foi determinar o efeito de 60 isolados de Trichoderma e do produto Trichodermil na promoção do crescimento e na indução de resistência sistêmica à antracnose, causada por Colletotrichum lagenarium em pepineiro, além de identificar as espécies dos isolados de Trichoderma spp. efetivas como indutores de resistência. Nos experimentos de promoção de crescimento, os isolados de Trichoderma spp. foram submetidos à inoculação no substrato e, após 21 dias, a massa de matéria seca da parte aérea das plantas foi mensurada. Nos experimentos de indução de resistência, os isolados que promoveram crescimento foram introduzidos no substrato, na base das plantas, sete dias antes da inoculação de C. lagenarium nas folhas. O isolado que apresentou melhor desempenho foi avaliado quanto à redução dos sintomas de antracnose, em aplicações aos 3, 7 ou 14 dias antes da inoculação do patógeno, e quanto à capacidade de aumentar a atividade de peroxidase. Dezenove isolados e o Trichodermil promoveram o crescimento de pepineiro em até 100% e conferiram proteção à antracnose em até 88,39%. O isolado IB 31/06 reduziu a severidade da doença nos intervalos de tempo avaliados. Não foi observado aumento significativo de peroxidase, sete dias após o tratamento com IB 31/06, nas plantas tratadas e infectadas com o patógeno, em comparação às plantas infectadas. O sequenciamento gênico dos dezenove isolados permitiu a identificação de sete espécies distintas de Trichoderma.

Design, Synthesis, Biological Activities, and 3D‐QSAR of New N,N′‐Diacylhydrazines Containing 2‐(2,4‐dichlorophenoxy)propane Moiety

A series of new N,N'-diacylhydrazine derivatives were synthesized efficiently under microwave irradiation. Their structures were characterized by (1) H NMR, MS, and elemental analysis. Various biological activities of these compounds were tested. Most of them exhibited higher herbicidal activities against dicotyledonous weeds than monocotyledonous weeds. In addition, favorable in vivo fungicidal activities were also found of these compounds against Cladosporium cucumerinum, Corynespora cassiicola, Sclerotinia sclerotiorum(Lib.)de Bary, Erysiphe cichoracearum, and Colletotrichum orbiculare (Berk aLMont) Arx. All compounds displayed excellent plant growth regulatory activities: 100% inhibition was achieved against the radicle growth of cucumber. To further investigate the structure-activity relationship, comparative molecular field analysis was performed on the basis of herbicidal activity data, resulting in a statistically reliable model with good predictive power (r(2) = 0.913, q(2) =0.556). Based on the calculation, five additional novel compounds were designed and synthesized. Satisfyingly, compound 4u displayed excellent herbicidal activity (94.7%) at 1500 g/ha, although it is less active than 2,4-D. Meanwhile, this compound also exhibited good fungicidal activity against C. orbiculare (Berk aLMont) Arx (82.16%).

Light and scanning electron microscopy studies on the infection process of melon leaves by Colletotrichum lagenarium

Colletotrichum lagenarium is the casual agent of anthracnose disease of melons. Light and scanning electron microscopy were used to observe the infection process of C. lagenarium on the leaves of two melon cultivars differing in susceptibility. On both cultivars conidia began germinating 12 h after inoculation (hai), forming appressoria directly or at the tips of germ-tubes. By 48 hai appressoria had melanised and direct penetration of host tissue had begun. On the susceptible cultivar, infection vesicles formed within 72 hai and developed thick, knotted primary hyphae within epidermal cells. By 96 hai C. lagenarium produced highly branched secondary hyphae that invaded underlying mesophyll cells. After 96 hai, light brown lesions appeared on the leaves, coincident with cell necrosis and invasion by secondary hyphae. While appressoria formed more quickly on the resistant cultivar, fewer germinated to develop biotrophic primary or invasive necrotrophic secondary hyphae than on the susceptible cultivar. These results confirm that C. lagenarium is a hemibiotrophic pathogen, and that resistance in melons restricts colonisation by inhibiting the development of necrotrophic secondary hyphae.

The role of a putative peroxisomal-targeted epoxide hydrolase of Nicotiana benthamiana in interactions with Colletotrichum destructivum, C. orbiculare or Pseudomonas syringae pv. tabaci

Motif analysis among 30 EH1 and EH2 epoxide hydrolases from Solanaceaeous plants showed differences primarily in the lid region around the catalytic site. Based on in silico models of 3D structures, EH1 proteins lack a catalytic triad because of the orientation of one of the conserved lid tyrosines, while the orientation of that tyrosine in EH2 proteins fomed a catalytic triad inside a hydrophobic tunnel. Two similar EH2 protein genes from Nicotiana benthamiana, NbEH2.1 and NbEH2.2, have a predicted peroxisomal targeting sequence, catalytic triad, and structural similarities to a potato cutin monomer-synthesizing epoxide hydrolase. NbEH2.1 expression increased with infections by the hemibiotrophs, Colletotrichum destructivum, Colletotrichum orbiculare or Pseudomonas syringae pv. tabaci only during their biotrophic phases, while there was only a slight increase during the hypersensitive response to P. syringae pv. tabaci ( avrPto). In contrast, among the four pathogens, NbEH2.2 expression increased only in response to P. syringae pv. tabaci. Virus-induced gene silencing of NbEH2.1 significantly affected only the interaction with C. destructivum, resulting in a delay in the appearance of necrosis that may be related to its biotrophic phase being restricted to single epidermal cells, which is unique among these pathogens. These results differed from that of a previously reported EH1 gene of N. benthamiana for these interactions, demonstrating specialization among EH genes in basal resistance.

The plant growth-promoting fungus Fusarium equiseti and the arbuscular mycorrhizal fungus Glomus mosseae stimulate plant growth and reduce severity of anthracnose and damping-off diseases in cucumber (Cucumis sativus) seedlings

To alleviate the environmental contamination due to persistent chemical usage, approaches to integrated pest management were conceived. In this perspective, microbe–microbe interactions such as mycorrhizal relationships with other soil microbiota in the rhizosphere like the plant growth-promoting fungi (PGPF) are particularly important. Better understanding of the interactions between beneficial microbial groups is imperative in the identification of possible synergistic or antagonistic effects to improve their practical usage as biocontrol agents or biofertilizers. In this study, the consequence of co-inoculation of the arbuscular mycorrhizal fungus (AMF) Glomus mosseae (Gm) and the PGPF Fusarium equiseti (isolates GF18-3 and GF19-1) in terms of plant growth enhancement, root and rhizosphere colonisation, and development of anthracnose (Colletotrichum orbiculare) and damping-off (Rhizoctonia solani AG-4) diseases in cucumber plants was investigated under controlled conditions. The amendment of either GF18-3 or GF19-1 singly or in combination with Gm indicated a general tendency to significantly enhance the shoot dry weight (SDW) of cucumber plants at 4 weeks after planting (WAP). Similarly, Gm alone significantly enhanced SDW at 4 WAP. Gm showed a tendency to depress root colonisation by F. equiseti but such antagonistic effect was not observed in the rhizosphere soil. Both GF18-3 and GF19-1 significantly reduced percent root colonisation of Gm. However, these general tendencies may vary with the inoculum densities of AMF and PGPF. Both F. equiseti and Gm inoculated singly significantly increased percent of protection against anthracnose, but the combined inoculation was more effective in controlling the disease compared to single inoculation. The inoculation of the cucumber seedlings with GF18-3, GF19-1 or Gm, 6 or 12 days prior to damping-off pathogen inoculation, increased percent of protection against damping-off disease. This study shows that the co-inoculation of F. equiseti and Gm resulted in additive effect on the suppression of anthracnose disease in cucumber.

Steroids and an oxylipin from an algicolous isolate of Aspergillus flavus

A new oxylipin, (8E,12Z)-10,11-dihydroxyoctadeca-8,12-dienoic acid (1), a new steroid, 3β,4α-dihydroxy-26-methoxyergosta-7,24(28)-dien-6-one (2), and four known steroids, episterol (3), (22E,24R)-ergosta-7,22-dien-3β,5α,6α-triol (4), (22E,24R)-ergosta-5,22-dien-3β-ol (5), and (22E,24R)-ergosta-4,6,8(14),22-tetraen-3-one (6), were isolated from the cultures of Aspergillus flavus, an endophytic fungus isolated from the marine red alga Corallina officinalis. Their structures and relative stereochemistry were elucidated by 1D, 2D NMR and mass spectroscopic techniques. 1 and 2 exhibited low activity to inhibit acetylcholinesterase and no activity against plant pathogenic fungi Colletotrichum lagenarium and Fusarium oxysporum.