Guanosine-specific single-stranded ribonuclease effectors of a phytopathogenic fungus potentiate host immune responses.

Plants activate immunity upon recognition of pathogen-associated molecular patterns. Although phytopathogens have evolved a set of effector proteins to counteract plant immunity, some effectors are perceived by hosts and induce immune responses. Here, we show that two secreted ribonuclease effectors, SRN1 and SRN2, encoded in a phytopathogenic fungus, Colletotrichum orbiculare, induce cell death in a signal peptide- and catalytic residue-dependent manner, when transiently expressed in Nicotiana benthamiana. The pervasive presence of SRN genes across Colletotrichum species suggested the conserved roles. Using a transient gene expression system in cucumber (Cucumis sativus), an original host of C. orbiculare, we show that SRN1 and SRN2 potentiate host pattern-triggered immunity responses. Consistent with this, C. orbiculare SRN1 and SRN2 deletion mutants exhibited increased virulence on the host. In vitro analysis revealed that SRN1 specifically cleaves single-stranded RNAs at guanosine, leaving a 3'-end phosphate. Importantly, the potentiation of C. sativus responses by SRN1 and SRN2, present in the apoplast, depends on ribonuclease catalytic residues. We propose that the pathogen-derived apoplastic guanosine-specific single-stranded endoribonucleases lead to immunity potentiation in plants.

First report of Colletotrichum orbiculare causing anthracnose of pointed gourd in India.

Pointed gourd (Trichosanthes dioica Roxb.), of the Cucurbitaceae family, is widely cultivated as a vegetable in many countries such as Bangladesh, India, Pakistan, Myanmar, Nepal and Sri Lanka. Over 800,000 metric tons of pointed gourds are produced annually in India, where cultivation is estimated to occupy over 33,000 hectares of land (MoA & FW, Government of India). In summer 2018, significant losses (approximately 15-20%) occurred in the sub-Himalayan region in West Bengal state of India (21.14-21.30° N, 78.82-79.02°E) due to a disease with typical anthracnose-like symptoms on the fruits. Light yellowish, small sized round to irregular spots were also apparent on the leaves. These spots gradually increased in size and turned into light brown and were surrounded by yellow halo. The lesions on the fruits were circular, yellow-brown, necrotic and sunken. A survey of four fields (1.5 ha) was conducted and a disease incidence of 30-40% was observed. Necrotic tissues from fruit as well as leaves were cut into approximately 5 mm2, surface sterilized with 0.1% HgCl2, plated in potato dextrose agar and incubated at 28ºC for 7 days in the dark. A total of 50 morphologically similar colonies were obtained from 20 sampled fruits and 10 sampled leaves. Fungal colonies were initially white, becoming gray as the cultures aged on PDA. The cultures developed black acervuli around the center of the colony. Setae were brown in colour, 1-5 septate, 40-100 µm long. Conidia were also observed through light and scanning electron microscopy and exhibit as (4-6 ×13-19 µm) hyaline, aseptate, cylindrical to oblong, with one end round and other truncate. The morphological characteristics were found similar to Colletotrichum orbiculare Damm, P.F. Cannon & Crous as reported by Damm et al. (2013). Ten isolates were obtained by transferring hyphal tips to new PDA plates and incubating under the same conditions. To confirm the identity of the pathogen, genomic DNA was extracted from five pure isolates (PG-Pha, PG-Pha-2, PG-Pha-3, PG-Pha-4, PG-Pha-5) with the cetyltrimethylammonium bromide (CTAB). Further, the ITS1-5.8S-ITS2 region, D1/D2 region of the 28S rRNA large subunit (LSU), Actin (ACT) gene and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were amplified using specific primers, ITS1/ITS4 (White et al. 1990), NL1/NL4, ACT1/ACT2 and GDF1/GDR1 respectively and PCR conditions described in Damm et al. (2012). A GenBank BLAST search showed 99-100% identity to the Colletotrichum orbiculare (Acc. Nos. KP898988 for ITS1-5.8S-ITS2, Z18997 for 28S rRNA, AB778553 for ACT gene, and KF178482 for GAPDH). All obtained sequences were submitted to the GenBank (Acc. Nos. MN006616, OP811046-OP811049, [ITS1-5.8-ITS2], MN006684, PP391616-PP391619 [28S rRNA], MN168524, PP400822-PP400825 [ACT gene], OP627091, PP400826-PP400829 [GAPDH]). For phylogenetic analysis, MEGA version 11 (Tamura et al. 2021) was used to construct a maximum likelihood tree with 1000 bootstrap replicates, based on a concatenation alignment of three gene sequences (ITS, Actin and GAPDH) of the all the five C. orbiculare isolates as well as sequences of other Colletotrichum species obtained from GenBank. The cluster analysis revealed that, isolate PG-Pha form a cluster with other C. orbiculare isolates. Pathogenicity tests were conducted to confirm Koch's postulates. Pathogenicity tests were performed in mature fruits by inoculating them (n=8) with 10 µl of a 1×106 conidia/ml suspension at needle puncture wound sites. In control set up sterile distilled water was pipetted on fruits. Fruits were placed on sterile trays covered with glasses and incubated at humid chambers at 28±2ºC with 12 h of light. Healthy one-month old potted pointed gourd plants (n=15) were sprayed with conidial suspension until run-off. A set of 15 plants were sprayed with sterile distilled water and maintained as control. The plants were kept in a greenhouse at 25ºC, >75% relative humidity, and a 16/8 h day/night cycle for 15 days. Sterile distilled water was sprayed on the plants at one day interval to maintain the humidity. Inoculated fruits started showing yellowing symptoms one day post inoculation and gradually yellow-brown sunken spots became visible at the place of puncture, whereas control fruits remain symptomless even after 7 days of inoculation. Inoculated leaves showed disease symptoms similar to those observed in the field whereas leaves of control sets were symptomless even after 15 days. The pathogenicity test was repeated thrice under the same conditions mentioned before. C. orbiculare was successfully re-isolated from all the symptomatic tissues of leaves as well as fruits, completing Koch's postulates. Previously, the pathogen has been reported as an important anthracnose pathogen of Cucurbitaceae, especially of cucumber (Cucumis sativus), melons (Cucumis melo), watermelon (Citrullus lanatus), pumpkin (Cucurbita pepo) and squash (Cucurbita maxima) (Farr and Rossman 2020). To our knowledge, this is the first report of C. orbiculare causing anthracnose of pointed gourd. This disease represents a threat to producers in India and central Asia. Further research may contribute to the development of management strategies for this disease.

The establishment of multiple knockout mutants of Colletotrichum orbiculare by CRISPR-Cas9 and Cre-loxP systems.

Colletotrichum orbiculare is employed as a model fungus to analyze molecular aspects of plant-fungus interactions. Although gene disruption via homologous recombination (HR) was established for C. orbiculare, this approach is laborious due to its low efficiency. Here we developed methods to generate multiple knockout mutants of C. orbiculare efficiently. We first found that CRISPR-Cas9 system massively promoted gene-targeting efficiency. By transiently introducing a CRISPR-Cas9 vector, more than 90% of obtained transformants were knockout mutants. Furthermore, we optimized a self-excision Cre-loxP marker recycling system for C. orbiculare because a limited availability of desired selective markers hampers sequential gene disruption. In this system, the integrated selective marker is removable from the genome via Cre recombinase driven by a xylose-inducible promoter, enabling the reuse of the same selective marker for the next transformation. Using our CRISPR-Cas9 and Cre-loxP systems, we attempted to identify functional sugar transporters involved in fungal virulence. Multiple disruptions of putative quinate transporter genes restricted fungal growth on media containing quinate as a sole carbon source, confirming their functionality as quinate transporters. However, our analyses showed that quinate acquisition was dispensable for infection to host plants. In addition, we successfully built mutations of 17 cellobiose transporter genes in a strain. From the data of knockout mutants that we established in this study, we inferred that repetitive rounds of gene disruption using CRISPR-Cas9 and Cre-loxP systems do not cause adverse effects on fungal virulence and growth. Therefore, these systems will be powerful tools to perform a systematic loss-of-function approach for C. orbiculare.

Selective deployment of virulence effectors correlates with host specificity in a fungal plant pathogen.

The hemibiotrophic fungal plant pathogen Colletotrichum orbiculare is predicted to secrete hundreds of effector proteins when the pathogen infects cucurbit crops, such as cucumber and melon, and tobacco (Nicotiana benthamiana), a distantly related Solanaceae species. Here, we report the identification of sets of C. orbiculare effector genes that are differentially required for fungal virulence to two phylogenetically distant host species. Through targeted gene knockout screening of C. orbiculare 'core' effector candidates defined based on in planta gene expression, we identified: four host-specific virulence effectors (named effector proteins for cucurbit infection, or EPCs) that are required for full virulence of C. orbiculare to cucurbit hosts, but not to the Solanaceae host N. benthamiana; and five host-nonspecific virulence effectors, which collectively contribute to fungal virulence to both hosts. During host infection, only a small subset of genes, including the host-specific EPC effector genes, showed preferential expression on one of the hosts, while gene expression profiles of the majority of other genes, including the five host-nonspecific effector genes, were common to both hosts. This work suggests that C. orbiculare adopts a host-specific effector deployment strategy, in addition to general host-blind virulence mechanisms, for adaptation to cucurbit hosts.

Fungal effector SIB1 of Colletotrichum orbiculare has unique structural features and can suppress plant immunity in Nicotiana benthamiana.

Fungal plant pathogens secrete virulence-related proteins, called effectors, to establish host infection; however, the details are not fully understood yet. Functional screening of effector candidates using Agrobacterium-mediated transient expression assay in Nicotiana benthamiana identified two virulence-related effectors, named SIB1 and SIB2 (Suppression of Immunity in N. benthamiana), of an anthracnose fungus Colletotrichum orbiculare, which infects both cucurbits and N. benthamiana. The Agrobacterium-mediated transient expression of SIB1 or SIB2 increased the susceptibility of N. benthamiana to C. orbiculare, which suggested these effectors can suppress immune responses in N. benthamiana. The presence of SIB1 and SIB2 homologs was found to be limited to the genus Colletotrichum. SIB1 suppressed both (i) the generation of reactive oxygen species triggered by two different pathogen-associated molecular patterns, chitin and flg22, and (ii) the cell death response triggered by the Phytophthora infestans INF1 elicitin in N. benthamiana. We determined the NMR-based structure of SIB1 to obtain its structural insights. The three-dimensional structure of SIB1 comprises five ß-strands, each containing three disulfide bonds. The overall conformation was found to be a cylindrical shape, such as the well-known antiparallel ß-barrel structure. However, the ß-strands were found to display a unique topology, one pair of these ß-strands formed a parallel ß-sheet. These results suggest that the effector SIB1 present in Colletotrichum fungi has unique structural features and can suppress pathogen-associated molecular pattern-triggered immunity in N. benthamiana.

CoGRIM19 is required for invasive hyphal growth of Colletotrichum orbiculare inside epidermal cells of cucumber cotyledons.

Colletotrichum orbiculare, an anthracnose disease fungus of cucurbit plants, extends penetration hyphae inside the epidermal cells of host plants. Unlike vegetative hyphae formed on a nutrient rich medium, this pathogen initially develops biotrophic penetration hyphae, which acquire nutrient resources from living host cells and secret effector proteins to suppress host defense responses. Subsequently, the nature of penetration hyphae changes from biotrophy to necrotrophy in response to the interaction with a host plant. Hence, controlling the extension of penetration hyphae is crucial for C. orbiculare infection. Here, we identified CoGRIM19 encoding Nadh-ubiquinone oxidoreductase subunit as a pathogenicity gene. Pathogenicity assays showed that the cogrim19 mutant caused no visible symptoms on cucumber cotyledons. Microscopic observations revealed that the cogrim19 mutant developed an appressorium and penetration hyphae under artificial conditions such as on coverslips or cellulose membranes, but the penetration hyphae of the mutant were retarded in the cucumber cotyledons. Microscopic observations of biotrophy-specific expression fluorescent signals revealed that the biotrophic stage was maintained in the retarded penetration hyphae of the cogrim19 mutant as the penetration of the wild type. In addition to cytological observations, pathogenicity assays using wounded leaves showed that the cogrim19 mutant had an attenuated pathogenesis. Taking our results together, CoGRIM19 is required for invasive hyphal growth inside the epidermal cells of cucumber cotyledons in C. orbiculare.

Reducing Chlorothalonil Use in Fungicide Spray Programs for Powdery Mildew, Anthracnose, and Gummy Stem Blight in Melons.

Fungicides are applied to nearly 80% of U.S. melon acreage to manage the numerous foliar and fruit diseases that threaten yield. Chlorothalonil is the most widely used fungicide but has been associated with negative effects on human and bee health. We designed alternative fungicide programs to examine the impact of reducing chlorothalonil use (Bravo Weather Stik) on watermelon, cantaloupe, and honeydew melon in 2016, 2017, and 2018 in Maryland. Chlorothalonil was replaced in the tank mix of weekly sprays of targeted fungicides with either polyoxin D zinc salt (Oso) or an extract of Reynoutria sachalinensis (Regalia). Powdery mildew (PM; Podosphaera xanthii), gummy stem blight (GSB; Stagonosporopsis spp.), and anthracnose (Colletotrichum orbiculare) were the most prevalent diseases to occur in the 3 years. Replacing chlorothalonil with the biopesticides as the tank-mix component of the fungicide spray program was successful in reducing GSB and PM severity in cantaloupe, honeydew melon, and watermelon compared with the untreated control, with the exception of GSB in 2017 in cantaloupe, and similar to the program including chlorothalonil in all cases, except anthracnose in watermelon. Anthracnose disease severity was not significantly reduced compared with the untreated control when chlorothalonil was replaced with the biopesticides and yields were not improved over the chlorothalonil-alone treatment in any of the trials. Therefore, replacement of chlorothalonil may not fully address its loss as a fungicide resistance management tool but efficacy can be maintained when polyoxin D is alternated with R. sachalinensis as a tank mix with targeted fungicides to manage PM and GSB.

Threonine synthase CoTHR4 is involved in infection-related morphogenesis during the pre-penetration stage in Colletotrichum orbiculare.

Upon recognition of host plants, Colletotrichum orbiculare, an anthracnose disease fungus of cucurbitaceous plants, initiates morphological differentiation, including conidial germination and appressorium formation on the cuticle layer. The series of infection processes of C. orbiculare requires enormous nutrient and energy, but the surface of the cucurbitaceous hosts is hardly nutrient-rich. Hence, C. orbiculare must exert tight management of its intracellular nutrients in order to properly induce infection-related morphogenesis. Here, we carried out a large-scale insertional mutagenesis screen using Agrobacterium tumefaciens-mediated transformation to identify novel genes involved in the pathogenicity of C. orbiculare and found that CoTHR4-encoded threonine synthase, a homolog of Saccharomyces cerevisiae THR4, is required for pathogenicity and conidiation in C. orbiculare. Threonine supplementation allowed the cothr4 mutant to produce conidia to a level equivalent to that of the wild-type. The conidia produced from the threonine-treated cothr4 mutant failed to germinate in the absence of threonine, but retained the ability to germinate and to form appressoria in the presence of threonine. However, the conidia produced from the threonine-treated cothr4 mutant remained attenuated in pathogenicity on cucumber cotyledons even in the presence of threonine. Cytorrhysis assays revealed that appressoria of the cothr4 mutant induced by exogenous threonine treatment showed low turgor generation. Taken together, these results showed that threonine synthase CoThr4 plays a pivotal role in infection-related morphogenesis during the pre-penetration stage of C. orbiculare.

Dysfunction of Arabidopsis MACPF domain protein activates programmed cell death via tryptophan metabolism in MAMP-triggered immunity.

Plant immune responses triggered upon recognition of microbe-associated molecular patterns (MAMPs) typically restrict pathogen growth without a host cell death response. We isolated two Arabidopsis mutants, derived from accession Col-0, that activated cell death upon inoculation with nonadapted fungal pathogens. Notably, the mutants triggered cell death also when treated with bacterial MAMPs such as flg22. Positional cloning identified NSL1 (Necrotic Spotted Lesion 1) as a responsible gene for the phenotype of the two mutants, whereas nsl1 mutations of the accession No-0 resulted in necrotic lesion formation without pathogen inoculation. NSL1 encodes a protein of unknown function containing a putative membrane-attack complex/perforin (MACPF) domain. The application of flg22 increased salicylic acid (SA) accumulation in the nsl1 plants derived from Col-0, while depletion of isochorismate synthase 1 repressed flg22-inducible lesion formation, indicating that elevated SA is needed for the cell death response. nsl1 plants of Col-0 responded to flg22 treatment with an RBOHD-dependent oxidative burst, but this response was dispensable for the nsl1-dependent cell death. Surprisingly, loss-of-function mutations in PEN2, involved in the metabolism of tryptophan (Trp)-derived indole glucosinolates, suppressed the flg22-induced and nsl1-dependent cell death. Moreover, the increased accumulation of SA in the nsl1 plants was abrogated by blocking Trp-derived secondary metabolite biosynthesis, whereas the nsl1-dependent hyperaccumulation of PEN2-dependent compounds was unaffected when the SA biosynthesis pathway was blocked. Collectively, these findings suggest that MAMP-triggered immunity activates a genetically programmed cell death in the absence of the functional MACPF domain protein NSL1 via Trp-derived secondary metabolite-mediated activation of the SA pathway.

A parasitic fungus employs mutated eIF4A to survive on rocaglate-synthesizing Aglaia plants.

Plants often generate secondary metabolites as defense mechanisms against parasites. Although some fungi may potentially overcome the barrier presented by antimicrobial compounds, only a limited number of examples and molecular mechanisms of resistance have been reported. Here, we found an Aglaia plant-parasitizing fungus that overcomes the toxicity of rocaglates, which are translation inhibitors synthesized by the plant, through an amino acid substitution in a eukaryotic translation initiation factor (eIF). De novo transcriptome assembly revealed that the fungus belongs to the Ophiocordyceps genus and that its eIF4A, a molecular target of rocaglates, harbors an amino acid substitution critical for rocaglate binding. Ribosome profiling harnessing a cucumber-infecting fungus, Colletotrichum orbiculare, demonstrated that the translational inhibitory effects of rocaglates were largely attenuated by the mutation found in the Aglaia parasite. The engineered C. orbiculare showed a survival advantage on cucumber plants with rocaglates. Our study exemplifies a plant-fungus tug-of-war centered on secondary metabolites produced by host plants.

Although plants may seem like passive creatures, they are in fact engaged in a constant battle against the parasitic fungi that attack them. To combat these fungal foes, plants produce small molecules that act like chemical weapons and kill the parasite. However, the fungi sometimes fight back, often by developing enzymes that can break down the deadly chemicals into harmless products. One class of anti-fungal molecules that has drawn great interest is rocaglates, as they show promise as treatments for cancer and COVID-19. Rocaglates are produced by plants in the Aglaia family and work by targeting the fungal molecule eIF4A which is fundamental for synthesizing proteins. Since proteins perform most of the chemistry necessary for life, one might think that rocaglates could ward off any fungus. But Chen et al. discovered there is in fact a species of fungi that can evade this powerful defense mechanism. After seeing this new-found fungal species successfully growing on Aglaia plants, Chen et al. set out to find how it is able to protect itself from rocoglates. Genetic analysis of the fungus revealed that its eIF4A contained a single mutation that 'blocked' rocaglates from interacting with it. Chen et al. confirmed this effect by engineering a second fungal species (which infects cucumber plants) so that its elF4A protein contained the mutation found in the new fungus. Fungi with the mutated eIF4A thrived on cucumber leaves treated with a chemical derived from rocaglates, whereas fungi with the non-mutated version were less successful. These results shed new light on the constant 'arms race' between plants and their fungal parasites, with each side evolving more sophisticated ways to overcome the other's defenses. Chen et al. hope that identifying the new rocaglate-resistant eIF4A mutation will help guide the development and use of any therapies based on rocaglates. Further work investigating how often the mutation occurs in humans will also be important for determining how effective these therapies will be.

Identification and Characterization of a Multifunctional Biocontrol Agent, Streptomyces griseorubiginosus LJS06, Against Cucumber Anthracnose.

Twenty-eight bacterial strains isolated from Chinese herb extracts, beer fermentation waste, and raw oyster shells were evaluated for their efficacy in controlling cucumber anthracnose. Four bacterial strains, namely TG01, TG02, LJS06, and LJS08, were found to effectively reduce the mycelial growth of Colletotrichum orbiculare COC3 on PDA media. Spraying or drenching LJS06 spore suspension before inoculation significantly p < 0.05 reduced disease severity; thus, LJS06 was subject to further characterization. On the basis of the morphological, physiological, and biochemical characteristics and a multilocus sequence analysis of partial 16S rRNA, atpD, rpoB, and trpB genes, LJS06 was identified to be Streptomyces griseorubiginosus (Ryabova and Preobrazhenskaya) Pridham et al. Physiological and biochemical tests revealed that S. griseorubiginosus LJS06 can produce amylase, cellulase, chitinase, protease, siderophore, polyamines, and indole-3-acetic acid. Thus, a culture filtrate of LJS06 (specifically SL06) was formulated and evaluated for its efficacy against conidial germination, appressorium formation, and anthracnose management. Diluted SL06 was found to significantly (p < 0.05) inhibit conidial germination and appressorium formation, which can be attributed to impaired membrane integrity, accumulated reactive oxygen species (ROS), and impaired energy metabolism in the conidia. In addition, the spraying and drenching of diluted SL06 before inoculation consistently and significantly (p < 0.05) reduced anthracnose severity. These results jointly suggest that S. griseorubiginosus LJS06 can aid in the management of cucumber anthracnose by directly inhibiting conidial function and priming the plant defense system.

Niemann-Pick Type C Proteins Are Required for Sterol Transport and Appressorium-Mediated Plant Penetration of Colletotrichum orbiculare.

Many biotrophic and hemibiotrophic fungal pathogens use appressoria to directly penetrate the host plant surface. In the cucumber anthracnose fungus Colletotrichum orbiculare, differentiation of appressoria requires a proper G1/S cell cycle progression, regulated by the GTPase-activating protein complex CoBub2-CoBfa1 and its downstream GTPase CoTem1. To explore the mechanisms by which the CoTem1 cascade regulates plant infection, we screened for CoTem1 interaction factors and identified a Niemann-Pick type C2 homolog (CoNpc2). Niemann-Pick type C proteins NPC1 and NPC2 are sterol-binding proteins required for sterol export from lysosomes (vacuoles) in humans and yeasts. We showed that CoNpc2 colocalized with CoNpc1 in late endosomes and vacuoles and that disruption of its gene resulted in aberrant sterol accumulation in vacuoles and loss of sterol membrane localization, indicating that NPC proteins are engaged in sterol transport in C. orbiculare. For appressorium infection, sterol transport and proper distribution mediated by CoNpc1 and CoNpc2 are critical for membrane integrity and membrane curvature with actin assembly, leading to penetration peg emergence and appressorial cone formation. Our results revealed a novel mechanism by which NPC proteins regulate appressorium-mediated plant infection. IMPORTANCE Fungal morphogenesis requires accurate cell cycle progression. Two-component GTPase-activating protein (GAP) CoBub2-CoBfa1 interacts with downstream GTPase CoTem1 and is required for G1/S progression to establish plant infection in Colletotrichum orbiculare. To understand the pathogenicity related functions of CoTem1 downstream, we identified a Niemann-Pick type C2 homolog (CoNpc2) as a novel physical interaction factor with CoTem1. Whereas NPC proteins (NPC1 and NPC2) are essential for sterol homeostasis in humans and yeasts, their functions in plant invasion by pathogenic fungi have remained unclear. In this study, we show that CoNPC1 and CoNPC2 play a critical role in intracellular sterol transport and that appropriate sterol distribution is required for membrane integrity and membrane curvature with actin assembly that leads to appressorium-mediated plant penetration and pathogenicity of C. orbiculare. Our findings suggest the importance of sterol distribution in fungal morphogenesis during plant infection.

Fungicide Formulations Influence Their Control Efficacy by Mediating Physicochemical Properties of Spray Dilutions and Their Interaction with Target Leaves.

In this study, three types of pyraclostrobin formulations (including emulsifiable concentrate (EC), suspension concentrate (SC), and microcapsules (MCs)) were used to control cucumber anthracnose. Pyraclostrobin EC had the highest inhibitory activity against Colletotrichum orbiculare in vitro. Much different from the bioactivity in vitro, pyraclostrobin MCs exhibited the highest control efficacy on cucumber anthracnose both in pot and field experiments. The physicochemical properties (particle size, surface tension) of the spray dilution, their interaction with target leaves (contact angle, adhesional tension, work of adhesion, retention, crystallization) and dissipation dynamic of the active ingredient were found to be highly potential factors that would significantly influence the control efficacy of pesticide formulations. Results showed that the control efficacies of different formulations of pyraclostrobin were determined mainly by the final behavior of the pesticides at the target interface, namely, the retention, crystallization, and dissipation dynamics of active ingredients. This study had revealed crucial factors that would influence the efficacy of different formulations of pyraclostrobin and thus could guide the rational and efficient use of different formulations of pesticides on target crops.

Induction of Systemic Resistance in Cucumber by Hypovirulent Binucleate Rhizoctonia against Anthracnose Caused by Colletotrichum orbiculare.

Treatment with hypovirulent binucleate Rhizoctonia (HBNR) isolates induced systemic resistance against anthracnose infected by Colletotrichum orbiculare in cucumber, as there were no direct interaction between HBNR and C. orbiculare. This is because of the different distances between HBNR and C. orbiculare, where the root was treated with HBNR isolate and C. orbiculare was challenged and inoculated in leaves or first true leaves were treated with HBNR isolate and C. orbiculare was challenged and inoculated in second true leaves. The use of barley grain inocula and culture filtrates of HBNR significantly reduced the lesion diameter compared to the control (p = 0.05). The total lesion diameter reduction by applying barley grain inoculum of HBNR L2, W1, W7, and Rhv7 was 28%, 44%, 39%, and 40%, respectively. Similar results was also observed in treatment using culture filtrate, and the reduction of total lesion diameter by culture filtrate of HBNR L2, W1, W7, and Rhv7 was 45%, 46%, 42%, and 48%, respectively. When cucumber root was treated with culture filtrates of HBNR, the lignin was enhanced at the pathogen penetration, which is spread along the epidermis tissue of cucumber hypocotyls. Peroxidase activity in hypocotyls in the treated cucumber plant with culture filtrates of HBNR significantly increased before and after inoculation of pathogens as compared to the control. Significant enhancement was also observed in the fast-moving anodic peroxidase isozymes in the treated plants with culture filtrates of HBNR. The results showed the elicitor(s) contained in culture filtrates in HBNR. The lignin deposition as well as the peroxidase activity is an important step to prevent systemically immunised plants from pathogen infection.

Establishment of a selection marker recycling system for sequential transformation of the plant-pathogenic fungus Colletotrichum orbiculare.

Genome sequencing of pathogenic fungi has revealed the presence of various effectors that aid pathogen invasion by the manipulation of plant immunity. Effectors are often individually dispensable because of duplication and functional redundancy as a result of the arms race between host plants and pathogens. To study effectors that have functional redundancy, multiple gene disruption is often required. However, the number of selection markers that can be used for gene targeting is limited. Here, we established a marker recycling system that allows the use of the same selection marker in successive transformations in the model fungal pathogen Colletotrichum orbiculare, a causal agent of anthracnose disease in plants belonging to the Cucurbitaceae. We identified two C. orbiculare homologues of yeast URA3/pyrG, designated as URA3A and URA3B, which can be used as selection markers on medium with no uridine. The gene can then be removed from the genome via homologous recombination when the fungus is grown in the presence of 5-fluoroorotic acid (5-FOA), a chemical that is converted into a toxin by URA3 activity. The ura3a/b double mutants showed auxotrophy for uridine and insensitivity to 5-FOA. Using the ura3a/b mutants, transformation with the URA3B marker and its removal were successfully applied to disrupt the virulence-related gene, PKS1. The pks1 mutants showed a reduction in virulence, demonstrating that the method can be used to study virulence-related genes in C. orbiculare. The establishment of a URA3-based marker recycling system in plant-pathogenic fungi enables the genetic analysis of multiple genes that have redundant functions, including effector genes.

Focal effector accumulation in a biotrophic interface at the primary invasion sites of Colletotrichum orbiculare in multiple susceptible plants.

We identified virulence-related effectors of a hemibiotrophic fungal pathogen Colletotrichum orbiculare, and found that a novel interface was generated by a biotrophic interaction between C. orbiculare and the host cucumber, in which the effectors secreted from the pathogen accumulated preferentially. The interface was located around the biotrophic primary hyphal neck. Here, we showed that C. orbiculare also developed this interface in a biotrophic interaction with melon, which belongs to Cucurbitaceae. Furthermore, C. orbiculare developed interface in the interaction with a susceptible plant, Nicotiana benthamiana, which is distantly related to Cucurbitaceae, suggesting that the spatial regulation strategy for effectors in C. orbiculare is not specific to cucumber; rather, it is conserved among the various plants that are susceptible to this pathogen.

Observations of Infection Structures after Inoculation with Colletotrichum orbiculare on the Leaves of Cucumber Plants Pre-inoculated with Two Bacterial Strains Pseudomonas putida or Micrococcus luteus.

Infection structures were observed at the penetration sites on the leaves of cucumber plants inoculated with Colletotrichum orbiculare using a fluorescence microscope. The cucumber plants were previously drenched with suspension of bacterial strains Pseudomonas putida or Micrococcus luteus. The plants pre-inoculated with both bacterial strains were resistant against anthracnose after inoculation with C. orbiculare. To investigate the resistance mechanism by both bacterial strains, the surface of infected leaves was observed at the different time after challenge inoculation. At 3 days after inoculation there were no differences in the germination and appressorium formation of conidia of C. orbiculare as well as in the callose formation of the plants between both bacteria pre-inoculated and non-treated. At 5 days, the germination and appressorium formation of the fungal conidia were, however, significantly decreased on the leaves of plants pre-inoculated with M. luteus at the concentration with 1.0 × 10(7) cfu/ml. Furthermore, callose formation of plants cells at the penetration sites was apparently increased. In contrast, there were no defense reactions of the plants at the concentration with 1.0 × 10(6) cfu/ml of M. luteus. Similarly, inoculation P. putida caused no plant resistance at the low concentration, whereas increase of callose formation was observed at the higher concentration. The results of this study suggest that the resistant mechanisms might be differently expressed by the concentration of pre-treatment with bacterial suspension.