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Strawberry fruit is highly appreciated worldwide for its organoleptic and healthy properties. However, this plant is attacked by many pathogenic fungi, which significantly affect fruit production and quality at pre- and post-harvest stages, making chemical applications the most effective but undesirable strategy to control diseases that has been found so far. Alternatively, genetic manipulation, employing plant key genes involved in defense, such as members of the NPR-like gene family, has been successful in many crops to improve resistance. The identification and use of the endogenous counterpart genes in the plant of interest (as it is the case of strawberry) is desirable as it would increase the favorable outcome and requires prior knowledge of their defense-related function. Using RNAi technology in strawberry, transient silencing of Fragaria ananassa NPR3 members in fruit significantly reduced tissue damage after Colletotrichum acutatum infection, whereas the ectopic expression of either FaNPR3.1 or FaNPR3.2 did not have an apparent effect. Furthermore, the ectopic expression of FaNPR3.2 in Arabidopsis thaliana double-mutant npr3npr4 reverted the disease resistance phenotype to Pseudomonas syringe to wild-type levels. Therefore, the results revealed that members of the strawberry FaNPR3 clade negatively regulate the defense response to pathogens, as do their Arabidopsis AtNPR3/AtNPR4 orthologs. Also, evidence was found showing that FaNPR3 members act in strawberry (F. ananassa) as positive regulators of WRKY genes, FaWRKY19 and FaWRKY24; additionally, in Arabidopsis, FaNPR3.2 negatively regulates its orthologous genes AtNPR3/AtNPR4. We report for the first time the functional characterization of FaNPR3 members in F. ananassa, which provides a relevant molecular basis for the improvement of resistance in this species through new breeding technologies.

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Red pepper powder (RPP) made from ground dried red pepper (Capsicum annuum L.) is prone to adulteration with fungal-spoiled RPP to gain unfair profits in Korea. This study aimed to investigate the effects of fungal infection on the ergosterol and phytosterol content of RPP and evaluate the potential of the sterol content as a marker for identifying fungal-spoiled RPP. Ergosterol was detected only in fungal-spoiled RPP and not in unspoiled RPP [

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Olive anthracnose is the most important fungal disease of the olive fruit worldwide, with the fungus Colletotrichum acutatum as the main cause of the disease in Greece. A total of 11 commercial biological plant protection products (bioPPPs) (Amylo-X®, Botector®, FytoSave®, LBG 01F34®, Mevalone®, Polyversum®, Remedier®, Serenade® ASO, Sonata®, Trianum-P®, Vacciplant®), with various modes of action against the fungus C. acutatum, were evaluated by bioassays using detached fruits of two important olive Greek varieties, cv. Koroneiki and cv. Kalamon. Subsequently, the most effective bioPPPs were evaluated for their ability to induce plant defense mechanisms, by determining the expression levels of ten Olea europaea defense genes (Pal, CuaO, Aldh1, Bglu, Mpol, Lox, Phely, CHI-2, PR-10, PR-5). Remedier®, Trianum-P®, Serenade® ASO, Sonata®, and Mevalone® were the most effective in reducing disease severity, and/or inhibiting the conidia production by the fungus at high rates. Post bioPPPs application, high expression levels of several olive plant defense genes were observed. This study provides insights into commercial bioPPPs' effectiveness in controlling olive anthracnose, as well as biocontrol-agents-mediated modulation of olive defense mechanisms.

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Agriculture currently confronts a multitude of challenges arising from the excessive utilization of chemical pesticides and the proliferation of phytopathogenic fungi strains that exhibit resistance to commonly employed active compounds in the field. Botrytis cinerea and Colletotrichum acutatum are phytopathogenic fungi that inflict substantial economic losses within agriculture and food due to their high impacts on crops both pre- and post-harvest. Furthermore, the emergence of fungal strains that are resistant to commercial fungicides has exacerbated this problem. To explore more environmentally sustainable alternatives for the control of these pathogens, an investigation into the endophytic bacteria associated with ginger (Zingiber officinale Rosc.) was conducted. The primary focus of this study involved evaluating their inhibitory efficacy against the fungi and assessing their potential for promoting plant growth. The endophytic bacteria genera Lelliottia, Lysinibacillus, Kocuria, Agrococcus, Acinetobacter, Agrobacterium, Zymobacter, and Mycolicibacterium were identified. All isolates showed remarkable in vitro antagonistic ability against B. cinerea (>94%) and C. acutatum (>74%). Notably, the Lelliottia amnigena J29 strain exhibited a notable proficiency in producing extracellular enzymes and indole compounds (IAA), solubilizing phosphate and potassium, and forming biofilm. Furthermore, the Lysinibacillus capsici J26, Agrococcus citreus J28, and Mycolicibacterium sp. J5 strains displayed the capacity for atmospheric nitrogen fixation and siderophore production. These findings underscore the agricultural and biotechnological potential of endophytic bacteria derived from ginger plants and suggest the feasibility of developing alternative approaches to manage these two phytopathogenic fungi.

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Anthracnose diseases, caused by Colletotrichum spp., are considered to be among the most destructive diseases that have a significant impact on the global production of strawberries. These diseases alone can cause up to 70% yield loss in North America. Colletotrichum spp. causes several disease symptoms on strawberry plants, including root, fruit, and crown rot, lesions on petioles and runners, and irregular black spots on the leaf. In many cases, a lower level of infection on foliage remains non-symptomatic (quiescent), posing a challenge to growers as these plants can be a significant source of inoculum for the fruiting field. Reliable detection methods for quiescent infection should play an important role in preventing infected plants' entry into the production system or guiding growers to take appropriate preventative measures to control the disease. This review aims to examine both conventional and emerging approaches for detecting anthracnose disease in the early stages of the disease cycle, with a focus on newly emerging techniques such as remote sensing, especially using unmanned aerial vehicles (UAV) equipped with multispectral sensors. Further, we focused on the acutatum species complex, including the latest taxonomy, the complex life cycle, and the epidemiology of the disease. Additionally, we highlighted the extensive spectrum of management techniques against anthracnose diseases on strawberries and their challenges, with a special focus on new emerging sustainable management techniques that can be utilized in organic strawberry systems.

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Strawberry (Fragaria x ananassa) production in Argentina extends to around 1700 hectares. Coronda city, located in Santa Fe province, is an important strawberry producer due to ideal agroecological conditions for culture and a high specialization for production. In November 2021, anthracnose symptoms were observed on strawberries cvs. 'San Andreas' and 'Splendor' in Coronda (31°58'S, 60°55'W), central Argentina. During these years, the incidence of the disease reached 40% of the production. Symptoms included 2-3 mm circular to irregular dark brown spots which enlarged rapidly and became sunken. Under high humidity conditions, concentric rings of pinhead-size salmon-colored acervuli developed on the lesions. The causal agent was isolated by touching acervuli with a sterile needle and monosporic cultures were obtained on PDA after 10 days at 25°C, with a 12-h light period. Colonies were white to gray on the top and orange on the underside, where concentric rings of salmon acervuli were clearly distinguished. The width and length of one hundred conidia were examined in three isolates (CF1, CF2, and CF3), ranging from 3.27 to 5.53 µm (avg.= 4.3 µm), and from 10.37 to 19.52 µm (avg.= 14.27 µm), respectively. The conidia were hyaline, smooth-walled, aseptate, and cylindric-clavate with one end round and one end acute. These morphological characteristics correspond to species belonging to the C. acutatum complex (Damm et al. 2012; Liu et al. 2022). To accurately identify the species, DNA was extracted from isolates, and ß-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and histone (HIS3) genes were partially amplified and sequenced (Vieira et al. 2020). TUB2, GAPDH, and HIS3 sequences presented a 100% of identity with species of Colletotrichum nymphaeae. The nucleotide sequences were deposited in GenBank (OR271556-OR271558, TUB2; OR271559-OR271561, GAPDH; and OR271562-OR271564, HIS3). Multilocus phylogenetic analyses performed with reference sequences (Damm et al. 2012) showed that the three isolates clustered with C. nymphaeae, in accordance with BLAST results. To confirm pathogenicity, each isolate was inoculated in eight detached fruits of the cultivar from which it was originally obtained. Two drops of 10 µl of conidial suspension (1x105 conidia per ml) were deposited in non-wounded areas on fruits previously disinfested with 1% sodium hypochlorite solution for 1 min and rinsed twice with sterile distilled water. Drops of sterile water were deposited in eight fruits as control. Pathogenicity tests were repeated twice. Fruits were kept in moist chamber (80+5% relative humidity) at 25°C for ten days. First symptoms appeared 4 days after inoculation. After that, all of the isolates produced symptoms identical to those previously described, whereas the controls remain symptomless. The pathogen was re-isolated from lesions, and identified as C. nymphaeae by morphological characteristics and based on the TUB2 sequences, as previously described. Strawberry anthracnose in Argentina was previously associated with Colletotrichum acutatum, C. gloeosporioides and C. fragariae species based on morphological characteristics (Ramallo et al. 2000; Monaco et al. 2000) but molecular identification was not performed until today. To our knowledge, this is the first report of C. nymphaeae causing anthracnose on strawberry in Argentina. This accurate identification will help to develop more efficient management strategies.

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Colletotrichum acutatum species complex is one of the most important groups in the genus Colletotrichum with a high species diversity and a wide range of host plants. C. acutatum and related species have been collected from different plants and locations in Korea and deposited into the Korean Agricultural Culture Collection (KACC), National Institute of Agricultural Sciences since the 1990s. These fungal isolates were previously identified based mainly on morphological characteristics, and a limitation of molecular data was provided. To confirm the identification of species, 64 C. acutatum species complex isolates in KACC were used in this study for DNA sequence analyses of six loci: nuclear ribosomal internal transcribed spacers (ITS), betatubulin 2 (TUB2), histone-3 (HIS3), glyceraldehyde3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), and actin (ACT). The molecular analysis revealed that they were identified in six different species of C. fioriniae (24 isolates), C. nymphaeae (21 isolates), C. scovillei (12 isolates), C. chrysanthemi (three isolates), C. lupini (two isolates), and C. godetiae (one isolate), and a novel species candidate. We compared the hosts of KACC isolates with "The List of Plant Diseases in Korea", previous reports in Korea and global reports and found that 23 combinations between hosts and pathogens could be newly reported in Korea after pathogenicity tests, and 12 of these have not been recorded in the world.

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Our study aimed to identify potential biocontrol agents (BCAs) against major phytopathogens under in vitro conditions by screening the Freshwater Bioresources Culture Collection (FBCC), Korea. Of the identified 856 strains, only 65 exhibited antagonistic activity, among which only one representative isolation, Brevibacillus halotolerans B-4359 was selected based on its in vitro antagonistic activity and enzyme production. Cell-free culture filtrate (CF) and volatile organic compounds (VOCs) of B-4359 were shown to be effective against the mycelial growth of Colletotrichum acutatum. Notably, B-4359 was found to promote spore germination in C. acutatum instead of exhibiting a suppressive effect when the bacterial suspension was mixed with the spore suspension of C. acutatum. However, B-4359 showed an excellent biological control effect on the anthracnose of red pepper fruits. Compared to other treatments and untreated control, B-4359 played a more effective role in controlling anthracnose disease under field conditions. The strain was identified as B. halotolerans using BIOLOG and 16S rDNA sequencing analyses. The genetic mechanism underlying the biocontrol traits of B-4359 was characterized using the whole-genome sequence of B-4359, which was closely compared with related strains. The whole-genome sequence of B-4359 consisted of 5,761,776 bp with a GC content of 41.0%, including 5,118 coding sequences, 117 tRNA, and 36 rRNA genes. The genomic analysis identified 23 putative secondary metabolite biosynthetic gene clusters. Our results provide a deep understanding of B-4359 as an effective biocontrol agent against red pepper anthracnose for sustainable agriculture.

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Anthracnose disease is a serious threat to red pepper crops in Korea and many other countries, resulting in considerable yield losses. There are now no effective control techniques available except for fungicide sprays, which may directly impact consumers. This study aims to investigate the biological activity of Trichoderma isolates in controlling red pepper anthracnose caused by Colletotrichum acutatum in vitro and in the field. Out of 11 Trichoderma isolates screened for biocontrol agents against three fungal pathogens, including C. acutatum; two effective Trichoderma isolates, T. atroviride ATR697 (ATR697) and T. longibrachiatum LON701 (LON701) were selected for further investigation. Using the overlapping plates experiment, it was discovered that the volatile organic compounds (VOCs) produced by ATR697 strongly inhibited C. acutatum mycelial growth to a larger extent than the isolate LON701. A cellophane membrane experiment has shown that mycelial growth of C. acutatum was inhibited by 36% and 27% when treated with ATR697 and LON701, respectively. Culture filtrates (CFs) of two Trichoderma isolates inhibited the mycelial growth of C. acutatum in vitro. When red peppers were treated with spore suspensions of LON701 and ATR697, the disease severity (%) was 44.1% and 55.8%, respectively, in a curative method; while the disease severity (%) was 5% and 11.6%, in LON701- and ATR697-treated red peppers, respectively, in a preventive method. These results showed the suppression of disease severity (%) was relatively higher in the preventive method than in the curative method. Furthermore, Trichoderma isolates ATR697 and LON701 were resistant to commercial chemical fungicides in vitro, indicating these strains may also be used synergistically with a chemical fungicide (pyraclostrobin) against the growth of C. acutatum. There was no difference in the inhibition rate (%) of the pathogen between the treatment with LON701 alone and LON701+pyraclostrobin. Based on in vitro findings, ATR697 and LON701 played a role in effectively controlling red pepper anthracnose in field conditions, with LON701 treatment resulting in a disease rate of 14% when compared to ATR697, chemical, and non-treated controls. Overall, our study showed the ability of Trichoderma isolates to control red pepper anthracnose and their potential to develop as novel biocontrol agents to replace chemical fungicides for eco-friendly, sustainable agriculture.

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Antimicrobial peptides (AMPs) from black solider flies (Hermetia illucens, BSF) exhibiting broad-spectrum antimicrobial activity are the most promising green substitutes for preventing the infection of phytopathogenic fungi; therefore, AMPs have been a focal topic of research. Recently, many studies have focused on the antibacterial activities of BSF AMPs against animal pathogens; however, currently, their antifungal activities against phytopathogenic fungi remain unclear. In this study, 7 AMPs selected from 34 predicted AMPs based on BSF metagenomics were artificially synthesized. When conidia from the hemibiotrophic phytopathogenic fungi Magnaporthe oryzae and Colletotrichum acutatum were treated with the selected AMPs, three selected AMPs-CAD1, CAD5, and CAD7-showed high appressorium formation inhibited by lengthened germ tubes. Additionally, the MIC50 concentrations of the inhibited appressorium formations were 40 µM, 43 µM, and 43 µM for M. oryzae, while 51 µM, 49 µM, and 44 µM were observed for C. acutatum, respectively. A tandem hybrid AMP named CAD-Con comprising CAD1, CAD5, and CAD7 significantly enhanced antifungal activities, and the MIC50 concentrations against M. oryzae and C. acutatum were 15 µM and 22 µM, respectively. In comparison with the wild type, they were both significantly reduced in terms of virulence when infection assays were performed using the treated conidia of M. oryzae or C. acutatum by CAD1, CAD5, CAD7, or CAD-Con. Meanwhile, their expression levels of CAD1, CAD5, and CAD7 could also be activated and significantly increased after the BSF larvae were treated with the conidia of M. oryzae or C. acutatum, respectively. To our knowledge, the antifungal activities of BSF AMPs against plant pathogenic fungi, which help us to seek potential AMPs with antifungal activities, provide proof of the effectiveness of green control strategies for crop production.

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The Colletotrichum acutatum species complex possesses a diverse number of important traits, such as a wide host range and host preference, different modes of reproduction, and different strategies of host infection. Research using comparative genomics has attempted to find correlations between these traits. Here, we used multi-locus techniques and gene genealogical concordance analysis to investigate the phylogenetic relationships and taxonomic status of the Colletotrichum acutatum species complex using field isolates obtained from rubber trees. The results revealed that the dominant species was C. australisinense, followed by C. bannaense, while strain YNJH17109 was identified as C. laticiphilum. The taxonomic status of strains YNLC510 and YNLC511 was undetermined. Using whole-genome single nucleotide polymorphism data to analyze population structure, 18 strains of C. australisinense were subsequently divided into four populations, one of which was derived from an admixture of two populations. In addition, the strains LD1687, GD1628, and YNLC516, did not belong to any populations, and were considered to be admixtures of two or more populations. A split decomposition network analysis also provided evidence for genetic recombination within Colletotrichum acutatum species complex from rubber trees in China. Overall, a weak phylogeographic sub-structure was observed. Analysis also revealed significant differences in morphological characters and levels of virulence between populations.

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In the current study we assessed a new crystallized compound, 5-(1-hydroxybutyl)-4-methoxy-3-methyl-2H-pyran-2-one (C-HMMP), from the endophytic fungus Colletotrichum acutatum residing in the medicinal plant Angelica sinensis for its in vitro antimicrobial, antibiofilm, antioxidant, antimalarial, and anti-proliferative properties. The promising compound was identified as C-HMMP through antimicrobial-guided fraction. The structure of C-HMMP was unambiguously confirmed by 2D NMR and HIRS spectroscopic analysis. Antimicrobial property testing of C-HMMP showed it to be effective against a variety of pathogenic bacteria and fungi with MICs ranging from 3.9 to 31.25 µg/ml. The compound displayed excellent antibiofilm activity against C. albicans, S. aureus, and K. pneumonia. Furthermore, the antimalarial and radical scavenging activities of C-HMMP were clearly dosede-pendent, with IC50 values of 0.15 and 131.2 µg/ml. The anti-proliferative activity of C-HMMP against the HepG-2, HeLa, and MCF-7 cell lines in vitro was investigated by MTT assay, revealing notable anti-proliferative activity with IC50 values of 114.1, 90, and 133.6 µg/ml, respectively. Moreover, C-HMMP successfully targets topoisomerase I and demonstrated beneficial anti-mutagenicity in the Ames test against the reactive carcinogenic mutagen, 2-aminofluorene (2-AF). Finally, the compound inhibited the activity of α-glucosidase and α-amylase with IC50 values of 144.7 and 118.6 µg/ml, respectively. To the best of our knowledge, the identified compound C-HMMP was obtained for the first time from C. acutatum of A. sinensis, and this study demonstrated that C-HMMP has relevant biological significance and could provide better therapeutic targets against disease.

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The genus Colletotrichum includes nine major clades with 252 species and 15 major phylogenetic lineages, also known as species complexes. Colletotrichum spp. are one of the top fungal plant pathogens causing anthracnose and pre- and postharvest fruit rots worldwide. Apple orchards are imperiled by devastating losses from apple bitter rot, ranging from 24 to 98%, which is a serious disease caused by several Colletotrichum species. Bitter rot is also a major postharvest rot disease, with C. fioriniae causing from 2 to 14% of unmarketable fruit in commercial apple storages. Dominant species causing apple bitter rot in the Mid-Atlantic United States are C. fioriniae from the Colletotrichum acutatum species complex and C. chrysophilum and C. noveboracense from the C. gloeosporioides species complex (CGSC). C. fioriniae is the dominant species causing apple bitter rot in the Northeastern and Mid-Atlantic states. C. chrysophilum was first identified on banana and cashew but has been recently found as the second most dominant species causing apple bitter rot in the Mid-Atlantic. As the third most dominant pathogen, C. noveboracense MB 836581 was identified as a novel species in the CGSC, causing apple bitter rot in the Mid-Atlantic. C. nupharicola is a sister group to C. fructicola and C. noveboracense, also causing bitter rot on apple. We deliver the resources of 10 new genomes, including two isolates of C. fioriniae, three isolates of C. chrysophilum, three isolates of C. noveboracense, and two isolates of C. nupharicola collected from apple fruit, yellow waterlily, and Juglans nigra. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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Biological control is a promising approach to reduce plant diseases caused by fungal pathogens and ensure high productivity in horticultural production. In the present study, we evaluated the biocontrol potential and underlying mechanisms of the beneficial fungus Aureobasidium pullulans against Botrytis cinerea and Colletotrichum acutatum, casual agents of gray mold and anthracnose diseases in strawberry. Notably, this is the first time that A. pullulans has been tested against C. acutatum in strawberry. A. pullulans strains (AP-30044, AP-30273, AP-53383, and AP-SLU6) showed significant variation in terms of growth and conidia production. An inverse relationship was found between the growth and conidiation rate, suggesting a trade-off between resource allocation for growth and conidial production. Dual plate co-culturing assays showed that mycelial growth of B. cinerea and C. acutatum was reduced by up to 35 and 18%, respectively, when challenged with A. pullulans compared with control treatments. Likewise, culture filtrates of A. pullulans showed varying levels of antifungal activity against B. cinerea and C. acutatum, reducing the mycelial biomass by up to 90 and 72%, respectively. Furthermore, milk powder plate assays showed that A. pullulans produced substantial amounts of extracellular proteases, which are known to degrade fungal cuticle. Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) analyses revealed that A. pullulans produced exophilins, liamocins, and free fatty acids known to have antifungal properties. A. pullulans shows high potential for successful biological control of strawberry diseases and discuss opportunities for further optimization of this beneficial fungus.

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Botrytis fruit rot (BFR) and anthracnose fruit rot (AFR) are diseases of concern to strawberry growers. Both diseases are managed mainly by fungicide applications from the nursery (plant production) to the end of the growing season (fruit production). In Florida, captan is the main broad-spectrum fungicide used to control BFR and AFR. It has been tested in many trials over the years in various programs in alternation with single-site fungicides or weekly applications. Due to its broad-spectrum activity, captan is a pivotal tool in fungicide resistance management, because resistance to several fungicides has been reported in populations causing BFR and AFR. Our objective was to determine the effectiveness and profitability of applications of captan for controlling BFR and AFR based on a univariate meta-analysis considering data from 25 field trials conducted from 2005 to 2021 in Florida. Captan applications significantly improved marketable yields and reduced BFR and AFR incidence during late- and total-season periods. Marketable yields were significantly improved even when the effect on disease control was not significant. Our results indicate 100% probability that weekly captan applications would return the investment during low, medium, and high strawberry pricing regimes, although the magnitude of the return will depend on strawberry market prices. However, the probabilities of reducing BFR, AFR, and culls were lower. Results from our meta-analysis demonstrate the value of captan as an important tool for strawberry growers. Captan applications, in addition to effectively controlling BFR and AFR and improving marketable yields, will result in investment returns at any strawberry price level.

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This study was aim at investigating antifungal activities of Bacillus velezensis FJAT-52631 and its lipopeptides against Colletotrichum acutatum ex situ and in situ. The results showed that the strain FJAT-52631 and its crude lipopeptides (10 mg/ml) exhibited strong inhibitory effects on growth of C. acutatum FJAT-30256 with an inhibition rate of 75.3% and an inhibition zone diameter of 17.66 mm, respectively. Both the viable bacterial cultures and lipopeptides of FJAT-52631 could delay the onset of loquat anthracnose by 1 day and lower the incidence of loquat anthracnose in situ. The whole cultures of B. velezensis FJAT-52631 displayed a 50% biocontrol efficacy on loquat anthracnose at the fourth day after inoculation, but the crude lipopeptides not. The average lesion diameter of the whole-culture treated group was 5.62 mm, which was smaller than that of control group (6.81 mm). All the three types of lipopeptides including iturin A, fengycin, and surfactin A secreted from the strain FJAT-52631 exhibited antifungal activities. Among them, surfactin A displayed higher antifungal activity at a concentration of 1.25 mg/mL than other two lipopeptides even if at a concentration of 60 mg/mL. Thus, the results indicated that surfactin A produced by FJAT-52631 played a major role in the biocontrol of the loquat anthracnose. Scanning electron microscopy (SEM) observation revealed the structural deformities in the mycelia of C. acutatum. The above results suggested that the antifungal lipopeptides from B. velezensis FJAT-52631 would be potential in biocontrol against anthracnose disease of loquat caused by C. acutatum.

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Colletotrichum acutatum, the main pathogen causing anthracnose on chili worldwide, is controlled by tebuconazole [a sterol C14-demethylation inhibitor (DMI) fungicide, abbreviated as Teb] with excellent efficacy. Our previous study exhibited that all C. acutatum isolates were sensitive to Teb while the Colletotrichum gloeosporioides population had developed resistance to Teb on the same fungicide-pressure selection. Therefore, the assessment of Teb-resistance in C. acutatum is impending. Twenty Teb-resistant (TebR) mutants obtained by fungicide domestication and ultraviolet (UV)-mutagenesis displayed similar fitness compared to parental isolates. Data in the current study exhibited that mutations at CaCYP51A and/or overexpression of CaCYP51s were responsible for Teb-resistance. Furthermore, the deletion mutants ΔCaCYP51A and ΔCaCYP51B played different roles in sensitivities to DMIs. Taken together, this study first reported that mutations at CaCYP51A and/or overexpression of CaCYP51s conferred resistance to Teb in C. acutatum, CaCYP51A and CaCYP51B are functionally redundant, but differentially regulated in DMI resistance.

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Colletotrichum australisinense, a member of the Colletotrichum acutatum species complex, is an important pathogen causing rubber tree anthracnose. Genome-wide comparative analysis showed this species complex contains more genes encoding necrosis- and ethylene-inducing peptide 1-like proteins (NLPs) than other Colletotrichum species complexes, but little is known about their necrosis-inducing roles in host. The aim of this study was to analyze NLPs number and type in C. australisinense, and characterize their necrosis-inducing activity in host or non-host. According to phylogenetic relationship, conserved the cysteine residues and the heptapeptide motif (GHRHDWE), 11 NLPs were identified and classified into three types. Five of the eleven NLPs were evaluated for necrosis-inducing activity. CaNLP4 (type 1) could not induce necrosis in host or non-host plants. By contrast, both CaNLP5 and CaNLP9 (type 1) induced necrosis in host and non-host plants, and necrosis-inducing activity was strongest for CaNLP9. CaNLP10 (type 2) and CaNLP11 (type 3) induced necrosis in host but not non-host plants. Substitution of key amino acid residues essential for necrosis induction activity led to loss of CaNLP4 activity. Structural characterization of CaNLP5 and CaNLP9 may explain differences in necrosis-inducing activity. We evaluated the expression of genes coding CaNLP by reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) at different time-points after pathogen infection. It was found that genes encoding CaNLPs with different activities exhibited significantly different expression patterns. The results demonstrate that CaNLPs are functionally and spatially distinct, and may play different but important roles in C. australisinense pathogenesis.

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Volatile organic compounds (VOCs) produced by microorganisms may prevent postharvest rot in fruits. Here, it was examined if VOCs from different species of Streptomyces can control infection in apples caused by the fungal pathogen Colletotrichum acutatum. Incubation of C. acutatum-infected apples in semi-closed boxes with actively growing strains of three Streptomyces (S. coelicolor, S. diastatochromogenes and Streptomyces strain 2R) showed that VOCs reduced rot areas of the apples by 45-66% after 8 days and 39-57% after 10 days, relative to infected apples incubated without Streptomyces. No differences in inhibition among the three strains were seen. In contrast, a mutant strain of Streptomyces that lacks major genes involved in biosynthesis of secondary metabolites, did not reduce development of rot in the apples. Furthermore, Streptomyces VOCs reduced radial hyphal growth of C. acutatum on agar. Several of the VOCs produced by three Streptomyces strains have previously shown fungicidal properties. Although the specific VOCs being active in inhibition of C. acutatum remain to be determined, VOCs may have a great potential as biofumigants to minimize postharvest diseases in fruits.

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The B. safensis RGM 2450 and B. siamensis RGM 2529 strains were isolated from the rhizosphere of plants presenting resilience to abiotic and biotic stress conditions. To understand the implications of bacteria in resilience, a genomic and experimental analysis was carried out on their biostimulant and phytopathogenic antagonist properties. Genome analyses of both strains indicated that they have the potential to synthesize bioactive compounds such as the battery of non-ribosomal peptides, polyketides, extracellular enzymes and phytohormones. These results were consistent with the antagonistic activities of both strains against the phytopathogens Botrytis cinerea, Colletotrichum acutatum, Fusarium oxysporum and Phytophtora cinnamomi. They also showed the capacity to solubilize phosphorus, fix nitrogen and produce indole acetic acid. This was observed in tomato seedlings grown from seeds inoculated with the mixture of strains which presented significantly greater length as well as wet and dry weight in comparison with the treatments individually inoculated with each strain and the control. Accordingly, the combination of B. safensis RGM 2450 and B. siamensis RGM 2529 showed synergistic biostimulant activity. These findings contribute new knowledge of the genomic and metabolomic properties taking part in the symbiotic interactions between these strains and the plants and uphold the combined use of both strains as a biostimulant.