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Cucurbit yellow vine disease (CYVD), which is caused by the gram-negative bacterium Serratia marcescens and transmitted by squash bugs ( Anasa tristis DeGeer), is a devastating disease of cucurbit crops that is emerging rapidly in the eastern half of the United States. The lack of a robust pathogenicity assay for CYVD in the laboratory has hampered functional tests using genomic sequences to investigate the biology of this phytopathogen. In this study we developed and validated a bioassay that yielded consistent and quantifiable CYVD symptoms on squash in the laboratory. We compared inoculation by wounding with a multipronged floral pin frog to inoculation by injection in which a needle was moved in and out of the stem multiple times in each of multiple piercings to mimic the feeding behavior of squash bugs. We found that inoculation by needle injection of ≥10 8 CFU/ml of S. marcescens into the stem of squash ( Cucurbita pepo ) plants at the cotyledon growth stage reproducibly induced CYVD symptoms, whereas injecting 10 6 or 10 7 CFU/ml did not. Additionally, we found that S. marcescens induced symptoms on all of the squash cultivars tested, and induced symptoms that have not been previously reported, including stem elongation and leaf cupping. In short, through our injection approach of mimicking the natural process of S. marcescens transmission by squash bug feeding, we obtained robust and quantifiable CYVD symptoms. This laboratory bioassay provides a crucial tool for investigating the biology and pathology of this emerging pathogen and for plant breeding screens aimed at combatting CYVD., Competing Interests: The author(s) declare no conflict of interest.

Potassium (K) fertilisation has frequently been shown to enhance plant resistance against pathogens, though the mechanisms remain elusive. This study investigates the interaction dynamics between Nicotiana benthamiana and the pathogen Alternaria longipes under different planta K levels. On the host side, adding K activated the expressions of three NLR (nucleotide-binding domain and leucine-rich repeat-containing proteins) resistance genes, including NbRPM1, NbR1B23 and NbNBS12. Silencing these NLRs attenuated resistance in high-K (HK, 40.8 g/kg) plant, whereas their overexpression strengthened resistance in low-K (LK, 23.9 g/kg) plant. Typically, these NLRs mainly strengthened plant resistance via promoting the expression of pathogenesis-related genes (PRs), ROS burst and synthesis of antifungal metabolites in HK plant. On the pathogen side, the expression of effectors HKCSP1, HKCSP2 and LKCSP were shown to be related to planta K content. A. longipes mainly expressed effectors HKCSP1 and HKCSP2 in HK plant to interfere host resistance. HKCSP1 physically interacted with NbRPM1 to promote the degradation of NbRPM1, then attenuated related resistance in HK N. benthamiana. Meanwhile, HKCSP2 directly interacted with NbPR5 to suppress resistance in HK plant. In LK plant, A. longipes mainly deployed LKCSP that interacted with NbR1B23 to interfere reduce resistance in N. benthamiana. Overall, our research insights that both pathogen and host mobilise distinct strategies to outcompete each other during interactions in different K nutrient environments., (© 2024 John Wiley & Sons Ltd.)

Colletotrichum fructicola shows morphological and genetic differences in plus and minus strains. However, the mechanism of the differentiation between two types of strains is still largely unclear. Our early transcriptome analysis revealed that CfHMG expression differed in plus and minus strains. To define the functions of the CfHMG gene, we constructed gene deletion mutants by homologous recombination. We found that a CfHMG deletion mutant of the minus strain, CfHMG-M, could lead to a reduction in perithecium sizes and densities on media and sterile perithecium formation compared with the minus wild type (WT), whereas there was no effect for the plus mutant CfHMG-P. In co-cultures between CfHMG-P and minus WT, CfHMG-M and plus WT, or CfHMG-P and CfHMG-M, the quantities of perithecia were all reduced significantly. When conidial suspensions were inoculated on non-wounded apple fruit, it was found that the virulence of the minus mutant decreased significantly but not for the plus one. Further, we found that the virulence decrease in minus mutants was caused by a decrease in the conidium germination rate. Our results indicate that CfHMG of C. fructicola plays an important role in the mating line formation between the plus and minus strain for both strains and differentially regulates the perithecium size, density, fertilization, and virulence of the minus strain. The results are significant for further detecting the differentiated mechanisms between the plus and minus strains in Colletotrichum fungi.

Cucurbit yellow vine disease (CYVD) is caused by Serratia marcescens , vectored by squash bugs ( Anasa tristis ), and is an emerging disease in many parts of the U.S. CYVD can cause 100% yield losses in cucurbits (Bruton et al., 2003). In the summer of 2021, at the Iowa State University Horticultural Research Station (HRS) in Gilbert, Iowa, we observed leaf yellowing, vine decline, and honey-brown discoloration of the phloem of acorn squash ( Cucurbita pepo cv. Table Ace) plants in research fields that were infested with squash bugs. In 2022, we observed similar symptoms on pumpkin ( Cucurbita maxima cv. Howden) and muskmelon ( Cucumis melo cv. Athena) in different fields at the HRS and on giant pumpkins ( Cucurbita maxima cv. Prizewinner) in Jones and Ringgold counties. For up to 3 symptomatic plants of each cucurbit species per location, a 20-cm-long stem section immediately above the soil line was excised, surface sterilized by immersion in 10% sodium hypochlorite and 70% ethanol for 2 min each, then triple rinsed in sterile water. The interior of the cross-section tissue was blotted on Luria agar amended with cycloheximide (100 μg/ml) and tetracycline (20 μg/ml) (Stock et al. 2003). Whitish translucent colonies developed after incubation at 28°C for 48 h. The genomic DNA of three isolates from symptomatic plants of muskmelon (MK01), pumpkin (HFP01), and giant pumpkin (AP01), was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD). S. marcescens species-specific primers YV1 (5'-GGGACTTGCTCCCCGG-3') and YV4 (5'-AACGTCAATTGATGAACGTATTAAGT-3') (Bruton et al. 2003) were used to amplify part of the 16S rDNA gene, and the primers specific to S. marcescens CYVD strains A79F/A79R (Zhang et al., 2005) were used to amplify part of a major facilitatory superfamily (MFS) transporter gene strain. The sequences of the 16S rRNA PCR product for the three isolates were identical and were deposited in NCBI under Accession OR963533. They shared 100% (395/395 nt) identity with other CYVD strains (Rascoe et al. 2003) and those of other S. marcescens strains in NCBI. The sequences of the amplified region of the MFS transporter gene of the three isolates (NCBI Accession OR962261) were identical and showed a 98.8% (319/323 nt) identity to that of non-CYVD-causing S. marcescens strains, such as N10A28 (Accession CP033623.1). Koch's postulates were fulfilled by inoculating C. pepo cv. Zephyr plants with either strain HFP01 or phosphate buffer saline (PBS) (10 plants per treatment) 1 wk after seeding by injecting 300 µl of bacteria (~10 8 CFU/ml) or PBS using a syringe needle. Plants were incubated at 28°C in a growth room for 4 wks. CYVD symptoms similar to those observed in the field developed on 7 out of 10 plants inoculated with strain HFP01 in one study, and 9 out 10 plants in a replicate study, with none of the PBS-inoculated plants showing CYVD symptoms. Bacteria were isolated from the symptomatic plants with selection on tetracycline. The PCR fragments amplified with YV1/YV4 and A79F/A79R were the same size as those of the pre-inoculation strain HFP01. To our knowledge this is the first report of CYVD in Iowa and in the Upper Midwest of the U.S. CYVD is a devastating disease that poses a significant threat to cucurbit production. This report can serve as an alert for the region's growers and for the development of effective management practices.

Fungal pathogens are one of the major reasons for biotic stress on rice (Oryza sativa L.), causing severe productivity losses every year. Breeding for host resistance is a mainstay of rice disease management, but conventional development of commercial resistant varieties is often slow. In contrast, the development of disease resistance by targeted genome manipulation has the potential to deliver resistant varieties more rapidly. The present study reports the first cloning of a synthetic maize chitinase 1 gene and its insertion in rice cv. (Basmati 385) via Agrobacterium-mediated transformation to confer resistance to the rice blast pathogen, Pyricularia oryzae. Several factors for transformation were optimized; we found that 4-week-old calli and an infection time of 15 minutes with Agrobacterium before colonization on co-cultivation media were the best-suited conditions. Moreover, 300 μM of acetosyringone in co-cultivation media for two days was exceptional in achieving the highest callus transformation frequency. Transgenic lines were analyzed using molecular and functional techniques. Successful integration of the gene into rice lines was confirmed by polymerase chain reaction with primer sets specific to chitinase and hpt genes. Furthermore, real-time PCR analysis of transformants indicated a strong association between transgene expression and elevated levels of resistance to rice blast. Functional validation of the integrated gene was performed by a detached leaf bioassay, which validated the efficacy of chitinase-mediated resistance in all transgenic Basmati 385 plants with variable levels of enhanced resistance against the P. oryzae. We concluded that overexpression of the maize chitinase 1 gene in Basmati 385 improved resistance against the pathogen. These findings will add new options to resistant germplasm resources for disease resistance breeding. The maize chitinase 1 gene demonstrated potential for genetic improvement of rice varieties against biotic stresses in future transformation programs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Colletotrichum fructicola is a devastating fungal pathogen of diverse plants. Sexually compatible plus and minus strains occur in the same ascus. However, the differentiation mechanism of plus and minus strains remains poorly understood. Here, we characterized a novel Cys2-His2-containing transcription factor CfCpmd1. The plus CfCpmd1 deletion mutant (Δ+ CfCpmd1 ) resulted in slow hyphal growth and a fluffy cotton-like colony, and the minus deletion mutant (Δ- CfCpmd1 ) exhibited characters similar to the wild type (WT). Δ+ CfCpmd1 led to defective perithecial formation, whereas Δ- CfCpmd1 produced more and smaller perithecia. The normal mating line was developed by pairing cultures of Δ- CfCpmd1 and plus WT, whereas a weak line was observed between Δ+ CfCpmd1 and minus WT. Conidial production was completely abolished in both plus and minus mutants. When inoculated on non-wounded apple leaves with mycelial plugs, Δ- CfCpmd1 was nonpathogenic because of failure to develop conidia and appressoria, while Δ+ CfCpmd1 could infect apple leaves by appressoria differentiated directly from hyphal tips, even though no conidia formed. Collectively, our results demonstrate that CfCpmd1 of C. fructicola is an important gene related to plus and minus strain differentiation, which also affects hyphal growth, sporulation, appressorium formation, and pathogenicity., Competing Interests: The author(s) declare no conflict of interest.

Cytospora canker, caused by Cytospora mali, is the most destructive disease in production of apples (Malus domestica). Adding potassium (K) to apple trees can effectively control this disease. However, the underlying mechanisms of apple resistance to C. mali under high-K (HK) status remain unknown. Here, we found that HK (9.30 g/kg) apple tissues exhibited high disease resistance. The resistance was impeded when blocking K channels, leading to susceptibility even under HK conditions. We detected a suite of resistance events in HK apple tissues, including upregulation of resistance genes, callose deposition, and formation of ligno-suberized tissues. Further multiomics revealed that the phenylpropanoid pathway was reprogrammed by increasing K content from low-K (LK, 4.30 g/kg) status, leading to increases of 18 antifungal chemicals. Among them, the physiological concentration of coumarin (1,2-benzopyrone) became sufficient to inhibit C. mali growth in HK tissues, and exogenous application could improve the C. mali resistance of LK apple branches. Transgenic apple calli overexpressing beta-glucosidase 40 (MdBGLU40), which encodes the enzyme for coumarin synthesis, contained higher levels of coumarin and exhibited high resistance to C. mali even under LK conditions. Conversely, the suppression of MdBGLU40 through RNAi reduced coumarin content and resistance in HK apple calli, supporting the importance of coumarin accumulation in vivo for apple resistance. Moreover, we found that the upregulation of transcription factor MdMYB1r1 directly activated MdBGLU40 and the binding affinity of MdMYB1r1 to the MdBGLU40 promoter increased in HK apple tissue, leading to high levels of coumarin and resistance in HK apple. Overall, we found that the accumulation of defensive metabolites strengthened resistance in apple when raising K from insufficient to optimal status, and these results highlight the optimization of K content in fertilization practices as a disease management strategy., Competing Interests: Conflict of interest statement. The authors have no conflict of interest in this study., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Training to enhance the effectiveness of oral presentations is often neglected in science, technology, engineering, and mathematics (STEM) fields. In this article, we summarize our experience of teaching a semester-long class in speaking skills to STEM graduate students and advocate for the critical importance of these skills to professional success., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Peach (Prunus persica) is an important stone fruit crop cultivated on 14,350 hectares in temperate areas of Pakistan, where post-harvest fungal rots cause major economic losses for growers. During April-August 2019, rotted peaches were observed in fruit markets of Rawalpindi district (33° 37' 33.8052'' N and 73° 4' 17.1912'' E), Punjab Province, Pakistan. Symptomatic fruit were surface sterilized for 3 min in 0.5% NaClO and 1 min in 70% ethanol, then rinsed three times in sterilized distilled water and air-dried for 30 minutes on filter paper. Tissue segments were excised from the margins of rot lesions and placed on potato dextrose agar (PDA), followed by incubation for one week at 25 +1o C. Furthermore, to maintain original sporodochial morphology and conidial description tissue segments were also grown on carnation leaf agar (CLA) and synthetic nutrient agar (SNA) (Sever et al., 2012). A total of 22 isolates were obtained from different fruit markets; colonies were fast-growing with pinkish-white, fluffy mycelia. Microconidia were abundant, 7.5 to 11.3 × 2.1 to 4.1 μm, oval to spindle-shaped, thin-walled, and hyaline whereas, macroconidia were thick-walled, sickle-shaped, 5- to 7-septate, hyaline, and 23.5 to 39.7 × 3.7 to 7.0 μm on carnation leaf agar. Virulence intensity of fungal isolates was confirmed after pathogenicity. Based on morphological characteristics, isolates were identified provisionally as Fusarium sporotrichioides (Leslie and Summerell 2006). For molecular identification, three target gene regions (ITS, elongation factor (EF), and β-tubulin benA) were amplified for highly pathogenic fungal isolates (FUS21K, FUS9SM, and FUS63KP) (O'Donnell et al. 2015). BLAST searches of the obtained sequences indicated 100% homology with GenBank accession number MN452643, and 99.56% homology with MT635298 for the ITS region, similarly 100% identity with GenBank accession numbers MN555016, MN555126, and MN555037 for the EF gene, and 100% homology with GenBank accession numbers MK435577, MH791363, and GQ915448 for β-tubulin benA of the F. sporotrichioides species complex (O'Donnell et al. 2015). Sequences were deposited in GenBank; accession numbers for each gene were as follows: ITS, ON180670, ON180671, and ON180672; EF, ON312093, ON312094, and ON312095; and β-tubulin benA, ON312090, ON312091, and ON312092. Maximum likelihood analysis on RPB1 and RPB2 nucleotide sequences of various fusaria was conducted which revealed that F. sporotrichioides formed a monophyletic relationship to terminal fusarium clade (TFC) comprising 22 strongly supported species complexes and 10 monotypic lineages, which were provisionally recognized as Fusarium. To complete Koch's postulates, 10μl aliquots of spore suspensions (106 spores/ml) of each FUS21K, FUS9SM and FUS63KP were pipetted individually onto six ripe, asymptomatic peaches. Sterile distilled water was applied as a negative control on four fruit. Fruit were incubated at 25 ± 1°C for one week in a sterile moist chamber, and the trial was conducted three times. Rot symptoms associated with pinkish-white fluffy mycelia were observed on inoculated fruits after 72 hours, whereas no mycelium was observed on the negative controls. The cultures on PDA, CLA and SNA from each of the inoculated fruit were morphologically identical to the original culture. The pathogen was confirmed as a member of F. sporotrichioides species complex based on pinkish-white mycelium, with red color on the underside of petri plates and the manner in which conidia are born on conidiophores. This is the first report of F. sporotrichioides on peaches from Pakistan, providing an important foundation for peach growers, agricultural advisers, plant pathologists, and plant breeders to improve the management of this economically damaging disease.

Glomerella leaf spot (GLS), caused by Colletotrichum fructicola , is a severe disease worldwide on apple, causing defoliation, leaf and fruit spot, and substantial yield loss. However, little is known about its molecular mechanisms of pathogenesis. Previous transcriptome analysis revealed that a transcription factor, CfMcm1, was induced during leaf infection. In the present work, expression pattern analysis verified that the CfMcm1 gene was strongly expressed in conidia and early infection. Phenotypic analysis revealed that the gene deletion mutant Δ CfMcm1 lost pathogenicity to apple leaves by inhibiting conidial germination and appressorium formation. In addition to appressorium-mediated pathogenicity, Δ CfMcm1 colonization and hyphal extension in wounded apple fruit was also reduced, and conidial germination mode and conidial color were altered. Δ CfMcm1 displayed impairment of cell wall integrity and response to stress caused by oxidation, osmosis, and an acid environment. Furthermore, the deletion mutant produced fewer and smaller perithecia and no ascospores. In contrast, melanin deposition in mycelia of Δ CfMcm1 was strengthened. Further comparative transcriptome and quantitative PCR analysis revealed that CfMcm1 modulated expression of genes related to conidial development ( CfERG5A , CfERG5B , CfHik5 , and CfAbaA ), appressorium formation ( CfCBP1 and CfCHS7 ), pectin degradation ( CfPelA and CfPelB ), sexual development ( CfMYB , CfFork , CfHMG , and CfMAT1-2-1 ), and melanin biosynthesis ( CfCmr1 , CfPKS1 , CfT4HR1 , CfTHR1 , and CfSCD1 ). Our results demonstrated that CfMcm1 is a pivotal regulator possessing multiple functions in pathogenicity, asexual and sexual reproduction, and melanin biosynthesis.

The predicted repertoire of type III secretion system effectors (T3SEs) in Erwinia tracheiphila , causal agent of cucurbit bacterial wilt, is much larger than in xylem pathogens in the closely related genera Erwinia and Pantoea . The genomes of strains BHKY and SCR3, which represent distinct E. tracheiphila clades, encode at least 6 clade-specific and 12 shared T3SEs. The strains expressed the majority of the T3SE genes examined in planta. Among the shared T3SE genes, eop1 was expressed most highly in both strains in squash ( Cucurbita pepo ) and muskmelon ( Cucumis melo ) but the clade-specific gene avrRpm2 was expressed 40- to 900-fold more than eop1 in BHKY. The T3SEs AvrRpm2, Eop1, SrfC, and DspE contributed to BHKY virulence on squash and muskmelon, as shown using combinatorial mutants involving six T3SEs, whereas OspG and AvrB4 contributed to BHKY virulence only on muskmelon, demonstrating host-specific virulence functions. Moreover, Eop1 was functionally redundant with AvrRpm2, SrfC, OspG, and AvrB4 in BHKY, and BHKY mutants lacking up to five effector genes showed similar virulence to mutants lacking only two genes. The T3SEs OspG, AvrB4, and DspE contributed additively to SCR3 virulence on muskmelon and were not functionally redundant with Eop1. Rather, loss of eop1 and avrB4 restored wild-type virulence to the avrB4 mutant, suggesting that Eop1 suppresses a functionally redundant effector in SCR3. These results highlight functional differences in effector inventories between two E. tracheiphila clades, provide the first evidence of OspG as a phytopathogen effector, and suggest that Eop1 may be a metaeffector influencing virulence. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Bitter rot and Glomerella leaf spot (GLS) of apples, caused by Colletotrichum species, are major diseases of apples around the world. A total of 98 isolates were obtained from apple fruits with bitter rot, and 53 isolates were obtained from leaves with leaf spot in the primary apple production regions in China. These isolates were characterized morphologically, and five gene regions ( ITS , ACT , GAPDH , CHS -1 and TUB 2) were sequenced for each isolate. A phylogenetic analysis, combined with a comparison of the morphological, cultural and pathogenic characters, sorted bitter rot isolates into six species: C. alienum , C. fructicola , C. gloeosporioides sensu stricto, C. nymphaeae , C. siamense and one new species, C. orientalis Dandan Fu & G.Y. Sun. Among these, C. siamense was the predominant pathogen associated with bitter rot. Isolates from leaf spot were identified as two species, C. aenigma and C. fructicola . This is the first report of C. orientalis as an apple bitter rot pathogen worldwide, and the results provide important insights into the diversity of Colletotrichum species in China.

Production of watermelon (Citrullus lanatus) in Malaysia was 150,000 mt in 2020 (Malaysian Department of Agriculture, 2021). In November 2019, nine locally produced watermelon fruit (red flesh, seedless) from five local stores in the states of Kelantan, Terengganu, and Penang exhibited sunken, circular, brown lesions that enlarged to1.5 to 10 cm in diameter with scattered orange masses of conidia. Lesions coalesced to cover approximately 50% of the fruit surface. Lesions were surface sterilized by spraying 70% alcohol onto the fruit followed by drying with sterilized paper towels. A total of 153 tissue segments (1×1 cm) were excised from the rind, immersed in 1% sodium hypochlorite for 3 min, rinsed twice for 1 min in sterilized distilled water, air-dried, transferred to potato dextrose agar (PDA) plates, and incubated at 25±1°C for 7 days. Single-spore transfers produced pure cultures, resulting in 12 isolates. Colonies on PDA were initially white and turned pale gray with age. Conidia were hyaline, one end round and the other narrowly acute, aseptate, smooth-walled, straight, cylindrical to clavate, 10.5-16.5 µm × 3-4.5 μm (n = 30). Observed morphological characters matched published description of Colletotrichum spp. (Damm et al. 2012). Internal transcribed spacer (ITS) and glyceraldehyde-phosphate dehydrogenase (GAPDH) genes were amplified using primer sets ITS1/ITS4 and GDF1/GDF2, respectively. All sequences were deposited in GenBank (MW856808 for ITS; MZ219296 for GAPDH). A BLASTn search of both sequences on GenBank showed 99% identity with C. scovillei along with other closely related Colletotrichum species. Phylogenetic analysis of ITS and GAPDH alignments, using maximum likelihood along with reference strains of closely related species from Mycobank, confirmed species identity as C. scovillei. A pathogenicity test was conducted on two healthy watermelon fruit (red flesh, seedless). A 6-mm-diameter mycelial plug of a colony on PDA was positioned on a 0.5-cm-long wound on each fruit; a sterile PDA plug placed on a similar wound on the opposite side served as a control. Fruit were incubated at 25±1°C for 7 days in plastic-wrapped trays above distilled water to maintain high humidity. Small, sunken, circular brown lesions appeared and expanded at inoculation sites within 7 days. Symptoms were identical to those produced by natural infections, and the controls were asymptomatic. Isolates from the lesions at the inoculation sites were confirmed as C. scovillei based on morphological characteristics, fulfilling Koch's postulates. The pathogenicity test was conducted four times with a total of eight fruit. Many species in the C. orbiculare complex cause watermelon anthracnose (Keinath, 2018). To our knowledge, this is the first report of C. scovillei (C. acutatum species complex; Damm et al. 2012) causing anthracnose on watermelon in Malaysia. Anthracnose caused by C. scovillei has been confirmed on other crops such as pepper (Toporek and Keinath, 2021), banana (Zhou et al., 2017), and chili (Oo et al., 2017). This insight will inform efforts to improve management of watermelon anthracnose in Malaysia.

Apple trees are grown worldwide, and consuming fresh apple fruit is associated with many health benefits. China produces about half of the world's apple supply. However, apple growing in China differs sharply from that in western countries in terms of the prevalent diseases and corresponding management strategies. For instance, family-owned small-scale orchards dominate China's apple industry, and manual bagging of fruit has been a long-standing practice for controlling fruit diseases. In recent years, rural labor shortages have been increasingly challenging the traditional production system, and China's apple industry is experiencing a rapid transition to much larger-scale enterprises featuring high-density orchards with advanced automation and mechanization. Associated with this transition are new challenges and grower demands that are changing the face of apple disease management. This Feature Article summarizes the ongoing transformation of China's apple industry in the context of sustainable disease management.

Strains of Erwinia tracheiphila, causal agent of bacterial wilt of cucurbits, are divided into distinct clades. Et-melo clade strains wilt Cucumis spp. but not Cucurbita spp., thus exhibiting host specificity, whereas Et-C1 clade strains wilt Cucurbita spp. more rapidly than Cucumis melo , thus exhibiting a host preference. This study investigated the contribution of the effector proteins Eop1 and DspE to E. tracheiphila pathogenicity and host adaptation. Loss of eop1 did not enable Et-melo strains to infect squash ( Cucurbita pepo ) or an Et-C1 strain to induce a more rapid wilt of muskmelon ( Cucumis melo ), indicating that Eop1 did not function in host specificity or preference as in the related pathogen E. amylovora. However, overexpression of eop1 from Et-melo strain MDCuke but not from Et-C1 strain BHKY increased the virulence of a BHKY eop1 deletion mutant on muskmelon, demonstrating that the Eop1 variants in the two clades are distinct in their virulence functions. Loss of dspE from Et-melo strains reduced but did not eliminate virulence on hosts muskmelon and cucumber, whereas loss of dspE from an Et-C1 strain eliminated pathogenicity on hosts squash, muskmelon, and cucumber. Thus, the centrality of DspE to virulence differs in the two clades. Et-melo mutants lacking the chaperone DspF exhibited similar virulence to mutants lacking DspE, indicating that DspF is the sole chaperone for DspE in E. tracheiphila , unlike in E. amylovora . Collectively, these results provide the first functional evaluation of effectors in E. tracheiphila and demonstrate clade-specific differences in the roles of Eop1 and DspE.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Botryosphaeria dothidea is one of the most common fungal pathogens on a large number of hosts worldwide. Botryosphaeria dothidea and B. kuwatsukai are also the main causal agents of apple ring rot. In this study, we sequenced, assembled and annotated the circular mitogenomes of 12 diverse B. dothidea isolates (105.7-114.8 kb) infecting various plants including apple, and five diverse B. kuwatsukai isolates (118.0-124.6 kb) from apple. B. dothidea mitogenomes harboured a set of 29-31 introns and 48-52 ORFs. In contrast, B. kuwatsukai mitogenomes harboured more introns (32-34) and ORFs (51-54). The variation in mitogenome sizes was associated mainly with different numbers of introns and insertions of mobile genetic elements. Interestingly, B. dothidea and B. kuwatsukai displayed distinct intron distribution patterns, with three intron loci showing presence/absence dynamics in each species. Large numbers of introns (57% in B. dothidea and 49% in B. kuwatsukai) were most likely obtained through horizontal transfer from non-Dothideomycetes. The mitochondrial gene phylogeny supported the differentiation of the two species. Overall, this study sheds light into the mitochondrial evolution of the plant pathogens B. dothidea and B. kuwatsukai, and intron distribution patterns could be useful markers for studies on population diversity., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Psidium guajava is a widely grown fruit tree of Asia for food and medicinal purposes. Also being reported to have anti-inflammatory, antimicrobial, antioxidant, antidiarrheal, antimutagenic properties (Somu, 2012). In April 2018, quick decline disease of guava was observed in orchards of Sheikhupura, Lahore, Faisalabad, Kasur and Chiniot districts of Punjab, Pakistan. Approximately 68% of the trees were found declined with mummified fruits. Initial infection symptoms appeared as wilting of leaves, bark discoloration, followed by the leaf drooping, crown area discoloration, bark splitting, mummified fruits, dying of branches and lately whole tree death in weeks to months. The fungus formed a dark brown to black discoloration (3 to 5 cm wide and 7 to 9 cm long) in vascular bundles of P. guajava tree. Sixty-five samples of discolored wood from the main stem were collected, and pathogen was isolated using carrot bait method (Moller and DeVay, 1968). Isolation and purification were done on 2% Malt extract agar (MEA) plates incubated at 25 ± 2 °C in 12 h light and dark period. After 6 days of incubation, fungal hyphae, fruiting structures, sexual & asexual spores were observed on MEA plates. Black globose to subglobose ascomata with bases (151-) 200 (-278) µm in diameter with long neck (511-) 535 to 600 (-671) µm long, (23-) 28 to 39 (-47) µm wide at base, (13-) 13- 19 (-25) µm wide at tip and light brown to hyaline divergent ostiolar hyphae (50µm) were developed and produces hat-shaped hyaline ascospores 3 to 5 µm long and 6-7 µm (with sheath) and 4 µm (without sheath) wide. After 7 days, initially white mycelium turned into olivaceous green and produced primary phialidic conidiophore with emerging primary cylindrical hyaline conidia (7 to 12 × 4 to 6 µm), secondary conidiophore with emerging chain of secondary barrel-shaped hyaline conidia (9-) 10 to 12 (-13) µm long × (5-) 5 to 9 (-11) µm wide and dark brown dematiaceous chlamydospores conidia (12 ×10 µm) were observed. All morphological characteristics were consistent to the description of Ceratocystis manginecans (Van Wyk, et al., 2007). For further confirmation, from a purified isolate GWD10, genomic DNA was extracted. The internal transcribed spacer (ITS) and translation elongation factor 1-alpha (TEF 1-α) region were amplified with primer pairs ITS1/ITS4 and EF1/EF2 (Jacobs et al., 2004; White et al., 1990) respectively. Generated sequences (Accession Nos. MN 365128 & MT952139) on BLAST analysis showed 100% homology for ITS and TEF with Ceratocystis manginecans (Accession No., KC261852 CMW 13582 Voucher, NR-119532.1 type material, MH863135; EF433317, respectively) reported from Oman and Pakistan (Van Wyk et al., 2007 & Vu et al., 2019). For pathogenicity test, one-year-old healthy P. guajava plants were inoculated by making a T-shaped slit of 5 × 7.5 mm in the bark. Two weeks old cultures of GWD10, 5-mm mycelial discs were aseptically transferred and covered with moistened sterilized cotton swab followed parafilm to maintain humidity. Fifteen plants were inoculated with fungal cultures and five plants were inoculated with MEA plugs as controls. All plants were maintained at 25 ± 2 °C with 80 ± 5% relative humidity (RH) in greenhouse Initial bark discoloration developed after 14 days of inoculation. After 40 days of inoculation plants started wilting and dying, similar to the symptoms were observed in naturally infected trees. Control plants remained asymptomatic. To fulfill Koch's pustulates, the same pathogen was re-isolated from the test plants and identified on morphological features to GWD10. The pathogen has been associated with mango decline in Oman and Pakistan (Van Wyk et al., 2007), acacia wilt in Indonesia (Harrington et al., 2015) and siris wilt in Pakistan (Razzaq et al., 2020). P guajava is an important fruit and medicinal plant, and the infection of C. manginecans is a great concern to the producers of P. guajava (Harrington et al., 2015; Huang et al., 2003). To our knowledge, this is the first report of Ceratocystis manginecans causing quick decline of P. guajava worldwide.

Glomerella leaf spot (GLS), caused by Colletotrichum fructicola , is a rapidly emerging disease leading to defoliation, fruit spot, and storage fruit rot on apple in China. Little is known about the mechanisms of GLS pathogenesis. Early transcriptome analysis revealed that expression of the zinc finger transcription factor Ste12 gene in C. fructicola ( CfSte12 ) was upregulated in appressoria and leaf infection. To investigate functions of CfSte12 during pathogenesis, we constructed gene deletion mutants (Δ CfSte12 ) by homologous recombination. Phenotypic analysis revealed that CfSte12 was involved in pathogenesis of nonwounded apple fruit and leaf, as well as wounded apple fruit. Subsequent histological studies revealed that loss of pathogenicity by Δ CfSte12 on apple leaf was expressed as defects of conidium germination, appressorium development, and appressorium-mediated penetration. Further RNA sequencing-based transcriptome comparison revealed that CfSte12 modulates the expression of genes related to appressorium function (e.g., genes for the tetraspanin PLS1, Gas1-like proteins, cutinases, and melanin biosynthesis) and candidate effectors likely involved in plant interaction. In sum, our results demonstrated that CfSte12 is a key regulator of early apple GLS pathogenesis in C. fructicola In addition, CfSte12 is also needed for sexual development of perithecia and ascospores. IMPORTANCE Glomerella leaf spot (GLS) is an emerging fungal disease of apple that causes huge economic losses in Asia, North America, and South America. The damage inflicted by GLS manifests in rapid necrosis of leaves, severe defoliation, and necrotic spot on the fruit surface. However, few studies have addressed mechanisms of GLS pathogenesis. In this study, we identified and characterized a key pathogenicity-related transcription factor, CfSte12, of Colletotrichum fructicola that contributes to GLS pathogenesis. We provide evidence that the CfSte12 protein regulates many important pathogenic processes of GLS, including conidium germination, appressorium formation, appressorium-mediated penetration, and colonization. CfSte12 also impacts development of structures needed for sexual reproduction which are vital for the GLS disease cycle. These results reveal a key pathogenicity-related transcription factor, CfSte12, in C. fructicola that causes GLS., (Copyright © 2020 American Society for Microbiology.)

The genus Rhodococcus contains several species with agricultural, biotechnological and ecological importance. Within this genus, many phyllosphere, rhizosphere and endosphere strains are plant growth promoting bacteria, whereas strains designated as R. fascians are plant pathogens. In this study, we isolated 47 Rhodococcus strains from a range of herbaceous and woody plant species. Phylogenetic analysis based on 16S rDNA, gyr B and alk B genes was used to compare our strains with type strains of Rhodococcus . For most of our strains, sequence similarity of the 16S rDNA, gyr B and alk B regions to type strains ranged from 98-100 %. Results of the concatenated gene sequence comparisons identified 18 strains of R. fascians and three strains of R. kroppenstedtii . The remaining strains were unclassified, and may represent novel species of Rhodococcus . Phylogenetic analysis based on gyr B sequences provided a more precise classification of our strains to species level than 16S rDNA sequences, whereas analysis of alk B sequences was unable to identify strains with orange-coloured colonies to species level.

Soybean rust (SBR), caused by the fungus Phakopsora pachyrhizi , is the most damaging disease of soybean in Brazil. Effective management is achieved by means of calendar-timed sprays of fungicide mixtures, which do not explicitly consider weather-associated disease risk. Two rain-based action thresholds of disease severity values (DSV50 and DSV80) were proposed and compared with two leaf wetness duration-temperature thresholds of daily values of infection probability (DVIP6 and DVIP9) and with a calendar program, with regard to performance and profitability. An unsprayed check treatment plot was included for calculating relative control. Disease severity and yield data were obtained from 29 experiments conducted at six sites across four states in Brazil during the 2012-13, 2014-15, and 2015-16 growing seasons, which represented different growing regions and climatic conditions. The less conservative rainfall action threshold (DSV80) resulted in fewer fungicide sprays compared with the other treatments, and the more conservative one (DSV50) resulted in fewer sprays than the DVIP thresholds. Yield was generally higher with the increase in spray number, but the economic analysis showed no significant differences in the risk of not offsetting the costs of fungicide sprays regardless of the system. Therefore, based on the simplicity and the profitability of the rain-based model, the system is a good candidate for incorporating into the management of SBR in soybean production fields in Brazil.

The spatial dissemination of three prevalent taxa of sooty blotch and flyspeck (SBFS) fungi under several levels of precipitation was compared during 2015 and 2016 in an Iowa apple orchard. Overhead irrigation was used to supplement ambient precipitation in order to insure SBFS spore dissemination and colony development. There were five irrigation levels, involving 1-min-long periods of irrigation that were imposed either once or twice per hour at intervals of 3, 6, or 12 h, as well as a nonirrigated control. Preselected apple fruit were inoculated with one of the three SBFS taxa to serve as sources of inoculum. Dissemination from these inoculated apple fruit was assessed at harvest by counting SBFS colonies on water-sprayed and nontreated fruit. As a further control, additional fruit were enclosed in fruit bags throughout the fruit development period. In both 2015 and 2016, the number of colonies of the SBFS fungus Peltaster gemmifer per apple increased sharply as the duration of irrigation increased, whereas the number of colonies of Microcyclosporella mali increased to a lesser extent and Stomiopeltis sp. RS1 showed no increase. In 2015, the linear relationship between the duration of irrigation-imposed precipitation levels and the number of colonies on the water-sprayed apple fruit was similar for P. gemmifer (slope = 0.09), Stomiopeltis sp. RS1 (slope = 0.07), and Microcyclosporella mali (slope = 0.13); whereas, in 2016, the slope was higher for P. gemmifer (0.28) than for Stomiopeltis sp. RS1 (-0.09) or M. mali (0.06). The results indicated that dissemination of P. gemmifer increased sharply in response to increased irrigation-imposed precipitation, and that dissemination patterns differed considerably among the three SBFS taxa. The apparent advantage of P. gemmifer in precipitation-triggered dissemination may stem from its ability to produce spores rapidly by budding. To our knowledge, this is the first article to assess splash dispersal by SBFS fungi in the field and the first to document taxon-specific patterns of dissemination in this pathogen complex.

Colletotrichum asianum is a worldwide plant pathogen causing serious fruit or leaf anthracnose diseases on a variety of plant hosts such as mango, coffee berry, chili, and other potential hosts, and it is distributed widely in Asia, America, Africa, and Oceania. This is the first genome resource available for C. asianum. The draft genome assembly will allow further analysis of species diversity and evolutionary mechanisms, and may serve as a foundation for genetic analysis that leads to greater understanding of interactions between plants and fungal pathogens.

The ascomycete fungus Colletotrichum fructicola causes diseases on a broad range of plant species. On susceptible cultivars of apple, it induces severe early defoliation and fruit spots, named glomerella leaf spot (GLS), but the mechanisms of pathogenicity have remained elusive. Phytopathogens exhibit small secreted effectors to advance host infection by manipulating host immune reactions. We report the identification and characterization of CfEC92, an effector required for C. fructicola virulence. CfEC92 is a Colletotrichum-specific small secreted protein that suppresses BAX-triggered cell death in Nicotiana benthamiana. Accumulation of the gene transcript was barely detectable in conidia or vegetative hyphae, but was highly up-regulated in appressoria formed during early apple leaf infection. Gene deletion mutants were not affected in vegetative growth, appressorium formation, or appressorium-mediated cellophane penetration. However, the mutants were significantly reduced in virulence toward apple leaves and fruits. Microscopic examination indicated that infection by the deletion mutants elicited elevated deposition of papillae at the penetration sites, and formation of infection vesicles and primary hyphae was retarded. Signal peptide activity, subcellular localization, and cell death-suppressive activity (without signal peptide) assays suggest that CfEC92 could be secreted and perform virulence functions inside plant cells. RNA sequencing and quantitative reverse transcription PCR results confirmed that the deletion mutants triggered elevated host defence reactions. Our results strongly support the interpretation that CfEC92 contributes to C. fructicola virulence as a plant immunity suppressor at the early infection phase., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Background: The wide variation in the size of fungal genomes is well known, but the reasons for this size variation are less certain. Here, we present a chromosome-scale assembly of ectophytic Peltaster fructicola, a surface-dwelling extremophile, based on long-read DNA sequencing technology, to assess possible mechanisms associated with genome compaction., Results: At 18.99 million bases (Mb), P. fructicola possesses one of the smallest known genomes sequence among filamentous fungi. The genome is highly compact relative to other fungi, with substantial reductions in repeat content, ribosomal DNA copies, tRNA gene quantity, and intron sizes, as well as intergenic lengths and the size of gene families. Transposons take up just 0.05% of the entire genome, and no full-length transposon was found. We concluded that reduced genome sizes in filamentous fungi such as P. fructicola, Taphrina deformans and Pneumocystis jirovecii occurred through reduction in ribosomal DNA copy number and reduced intron sizes. These dual mechanisms contrast with genome reduction in the yeast fungus Saccharomyces cerevisiae, whose small and compact genome is associated solely with intron loss., Conclusions: Our results reveal a unique genomic compaction architecture of filamentous fungi inhabiting plant surfaces, and broaden the understanding of the mechanisms associated with compaction of fungal genomes.

The El Niño Southern Oscillation (ENSO) is an oceanic-atmospheric phenomenon influencing worldwide weather and climate. Its occurrence is determined by the sea surface temperature (SST) anomaly of the 3.4 Niño region in the Pacific Ocean (5°N-5°S, 120°-170°W). El Niño (EN), Neutral (NT), and La Niña (LN) are the three possible phases of ENSO, respectively, for warm, normal, and cold SST anomaly. As in other regions around the world, weather in Brazil is influenced by ENSO phases. The country is the major coffee producer in the world, and production is strongly influenced by weather conditions, which affect plant yield, harvest quality, and interactions with pests and diseases. Coffee leaf rust (CLR), caused by the fungus Hemileia vastatrix , is a major cause of coffee yield and quality losses in Brazil, and requires fungicide spray applications every season. Because CLR is highly influenced by weather conditions, it is possible to use weather variables to simulate its progress during the cropping cycle. Therefore, the aims of this study were to estimate CLR infection rate based on a validated empirical model, which has daily minimum air temperature and relative humidity as inputs, and to assess the extent of ENSO influence on the annual risk of this disease at 45 sites in Brazil. Cumulative infection rates (CIR) were estimated daily from October to June of each growing season and location, based on the prevailing ENSO phase. Differences between the extreme phases (EN-LN) were assessed by the Two-One-Sided-Tests (TOST) method. Analysis of data from eight sites, located mainly in Paraná State, provided evidence of CIR differences between EN and LN phases (G1). Evidence of no difference of CIR between EN and LN was found in 18 sites (G2), whereas 19 sites showed no evidence of differences (G3) due to relatively large variation of CIR within the same ENSO phase. The G1 sites are located mostly in Southern Brazil, where ENSO exerts a well-defined influence on rainfall regime. In contrast, the G2 sites are mainly in Minas Gerais State, which is characterized as a transition region for ENSO influence on rainfall. The G3 sites are located between the northern region of Minas Gerais State and southern region of Bahia State, which is characterized by a subhumid climate that is usually very dry during winter, and where rainfall can vary up to 300% from one year to another, influencing relative humidity and resulting in a high CIR variability. Therefore, ENSO had a well-defined influence on CIR only in Paraná State, a region with minor importance for coffee production in Brazil. No ENSO influence was found in more northerly zones where the majority of Brazilian coffee is produced. This is the first evidence of ENSO-linked regional impact on the risk of coffee rust.

Apple core rot, an economically important disease worldwide, appears both before and during harvest. Current gaps in understanding of the infection cycle impede progress toward more effective management of this disease. The fungus Trichothecium roseum is the main pathogen of core rot on apple in China. In this study, we used fluorescent labeling to trace colonization of T. roseum in floral tissues, characterizing routes of penetration to the core of 'Fuji' apples. T. roseum infected petals, anthers, filaments, stigmas and separated styles of flowers, and floral debris served as inoculum for core infection. In field inoculations, T. roseum entered styles initially through stylar fissures and colonized pluricellular hairs of these fissures during early stages of fruit development. Subsequently, hyphae grew along the extending fissures, which are continuations of stylar fissures located between stylar bases and carpel cavities. The hyphae remained in the extending fissures from mid-June to late July. When fruit developed an open sinus in late July, the sinus eventually fused with extending fissures and carpel cavities in late August, hyphae invaded carpel cavities, and ultimately fruit flesh via cracks on carpel cavity walls. Our results revealed for the first time the routes by which T. roseum penetrates apple fruit, and provided significant insights for strategic management of core rot.

The objectives of this study were to evaluate the efficacy of integrating resistant genotypes of Jerusalem artichoke with Trichoderma harzianum isolate T9 to control Alternaria leaf spot caused by Alternaria spp. under two fertilization regimes and to determine whether T9 application induced chitinase and β-1,3-glucanase activity in Jerusalem artichoke leaves. Six Jerusalem artichoke varieties (resistant varieties JA15, JA86, and JA116 and susceptible varieties HEL246, HEL293, and JA109) and three disease control methods (a non-inoculated control, application of T. harzianum T9, and fungicide sprays (propiconazole at a rate of 30 mL/20 L of water, 375 ppm)) was conducted in two separate trials (different fertilization regimes) at the experimental farm of the Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand. Resistant genotypes controlled Alternaria leaf spot effectively. Application of Trichoderma showed low efficacy to control Alternaria leaf spot, but in specific susceptible genotypes-HEL246 and HEL293-the application of Trichoderma could reduce disease severity up to 10%. The application of Trichoderma was associated with a rise in production of chitinase and β-1,3-glucanase in HEL246 seedlings. The number of Trichoderma propagules in soil, as well as the extent of colonization of roots and leaves, were monitored. The results indicated that application of Trichoderma had higher propagules than non-inoculated control. Neither varietal resistance nor the disease control methods used in this study impacted the yield or yield components of Jerusalem artichoke.

Sooty blotch and flyspeck (SBFS) fungi produce superficial, dark-colored colonies on fruits, stems, and leaves of many plant genera. These blemishes are economically damaging on fruit, primarily apple and pear, because they reduce the sale price of fresh fruit. Fungicide spray programs can control SBFS but are costly and impair human and environmental health; thus, less chemically intensive management strategies are needed. Although the scientific study of SBFS fungi began nearly 200 years ago, recent DNA-driven studies revealed an unexpectedly diverse complex: more than 100 species in 30 genera of Ascomycota and Basidiomycota. Analysis of evolutionary phylogenetics and phylogenomics indicates that the evolution of SBFS fungi from plant-penetrating ancestors to noninvasive ectophytic parasites was accompanied by a massive contraction of pathogenicity-related genes, including plant cell wall-degrading enzymes and effectors, and an expansion of cuticle-degradation genes. This article reviews progress in understanding SBFS taxonomy and ecology and improving disease management. We also highlight recent breakthroughs in reconstructing the evolutionary origins of these unusual plant pathogens and delineating adaptations to their ectophytic niche.

Apple fruit spot disease has caused serious economic losses for years in China since the widespread application of fruit bagging in production. Although the three genera Trichothecium , Alternaria , and Acremonium have been reported to be the causal agents, studies on the disease etiology and pathogen biology are still sparse. Here, we report characterization of eight fungal isolates from lesions on 126 symptomatic fruit samples collected in Shaanxi Province, China. Pathogenicity of the isolates was assessed. DNA sequences were obtained at four loci, including D1/D2 domains of the large-subunit nrRNA gene, internal transcribed spacer regions 1 and 2, 5.8S nrDAN gene, a fragment of the actin gene, and a fragment of the β-tubulin. Based on phylogenetic analysis and morphological features, three new species were found: Acremonium mali , Sarocladium liquanensis , and Sarocladium mali . In addition, we made the first report of Sarocladium terricola as a plant pathogen. Temperature and moisture significantly affected in vitro conidial germination of five Acremonium -like species, and their impact on infection of apple fruit was tested using Acremonium sclerotigenum . Conidia of five species germinated from 15 to 35°C in free water; four of the species had optimum temperature around 25°C, whereas conidia of S. terricola had an optimum temperature of 30°C. Conidial germination rate increased as relative humidity (RH) increased. The five isolates had relatively high conidial germination rates at RH > 97%, with a significant decline at 95% RH. Incidence of infection also increased in proportion to RH. In free water, conidial germination was relatively unaffected by temperature.

Colletotrichum siamense causes fruit or foliar disease called anthracnose on a variety of plant hosts such as vegetables, fruits, ornamental plants, and others, including chili pepper, apple, American cranberry, mango, orange, papaya, guava, rubber plant, jasmine, coffee berry, and tea plants. Here, we report the first Illumina-sequenced draft genome assembly of C. siamense strain ICMP 18578 and its annotation. This genome sequence provides a unique resource that will be useful for future research on the evolution of Colletotrichum spp. and improvement of anthracnose management strategies.

Fruit bagging is a widely used orchard practice in China. Trichothecium black spot (TBS) is a disease highly associated with the fruit bagging. In this study, we characterized in vitro factors affecting the causal agent, Trichothecium roseum, and TBS development and infection histology on field-bagged apple fruit in situ. Under in vitro conditions, conidial germination required exogenous nutrients, and the germination rate was significantly promoted by high humidity, a condition mimicking the bag microenvironment. Germ tubes penetrated fruit via natural openings including stomata, lenticels, and surface cracks. To determine the chronology of infection by T. roseum, 'Fuji' fruit were inoculated in the field at different developmental stages. The earliest infection occurred 60 days after full bloom (dafb), and disease incidence increased as fruit maturity advanced. At harvest time (165 dafb), lesions on more recently inoculated fruit (105 dafb, 150 dafb) were larger than lesions from fruit inoculated on earlier dates. Histological observation showed that infection of younger fruit elicited stronger host lignification responses restricting lesion development. Taken together, our results support the hypothesis that high humidity in sealed bags and increased susceptibility associated with advancing fruit maturity are key factors promoting T. roseum infection and TBS symptom development on bagged apple fruit.

Field validation of a disease-warning system for strawberry anthracnose fruit rot (AFR), caused by Colletotrichum acutatum sensu lato, that was originally developed for use in Florida was conducted in Iowa from 2012 to 2014. Day-neutral strawberry (cv. Tristar) was artificially inoculated with the pathogen at the start of the fruit maturation period. A factorial combination in replicated trials of two spray timing methods (the Florida warning system and prescheduled, calendar-based timing) and two fungicides (captan and pyraclostrobin) was compared, along with a nonsprayed control. The calendar-based and warning system-based treatments provided statistically equivalent control of AFR incidence compared with the nonsprayed control, and the warning system treatments required an average of 1.7 fewer fungicide sprays annually than the calendar-based treatments. Further analysis of the field data suggested that the warning system might underestimate AFR risk under high disease pressure; in these circumstances, a lower action threshold value may need to be adopted. Overall, these results indicated that the Florida warning system can be valuable for helping Midwest strawberry growers control AFR with less reliance on fungicide sprays, but it may require modification to account for periods of high inoculum pressure, subject to results of further field trials.

Sooty blotch and flyspeck (SBFS) fungi infect the cuticle of fruit, including apple fruit, and produce pigmented colonies. A new member of this fungal complex in the genus Peltaster is described on the basis of molecular and morphological evidence. The SBFS complex is a diverse group of ectophytic fungi that reside primarily within the order Capnodiales. Sooty blotch and flyspeck isolates from apple orchards in the central United States were subjected to parsimony and Bayesian analyses based on the internal transcribed spacer regions of nuc rDNA, the partial translation elongation factor 1-α gene, and the partial mitochondrial small subunit rRNA gene. Phylogenetic analysis delineated a new species, Peltaster gemmifer, from P. cerophilus and P. fructicola. Peltaster gemmifer conidiophores bear primary conidia that produce secondary conidia either through budding or through microcyclic conidiation; these were not seen in cultures of P. cerophilus and P. fructicola. On cellulose membrane that was placed on water agar amended with apple juice, P. gemmifer produced brown to black pycnothyria in a superficial brownish mycelial mat, similar to the colonies produced on apple fruit. Findings from the present study add to the >80 named and putative SBFS species so far described worldwide.

Background: Colletotrichum is a fungal genus in Ascomycota that contain many plant pathogens. Among all Colletotrichum genomes that have been sequenced, C. fructicola contains the largest number of candidate virulence factors, such as plant cell wall degrading enzymes, secondary metabolite (SM) biosynthetic enzymes, secreted proteinases, and small secreted proteins. Systematic analysis of the expressional patterns of these factors would be an important step toward identifying key virulence determinants., Results: In this study, we obtained and compared the global transcriptome profiles of four types of infection-related structures: conidia, appressoria, infected apple leaves, and cellophane infectious hyphae (bulbous hyphae spreading inside cellophane) of C. fructicola. We also compared the expression changes of candidate virulence factors among these structures in a systematic manner. A total of 3189 genes were differentially expressed in at least one pairwise comparison. Genes showing in planta-specific expressional upregulations were enriched with small secreted proteins (SSPs), cytochrome P450s, carbohydrate-active enzymes (CAZYs) and secondary metabolite (SM) synthetases, and included homologs of several known candidate effectors and one SM gene cluster specific to the Colletotrichum genus. In conidia, tens of genes functioning in triacylglycerol biosynthesis showed coordinately expressional upregulation, supporting the viewpoint that C. fructicola builds up lipid droplets as energy reserves. Several phosphate starvation responsive genes were coordinately up-regulated during early plant colonization, indicating a phosphate-limited in planta environment immediately faced by biotrophic infectious hyphae., Conclusion: This study systematically analyzes the expression patterns of candidate virulence genes, and reveals biological activities related to the development of several infection-related structures of C. fructicola. Our findings lay a foundation for further dissecting infection mechanisms in Colletotrichum and identifying disease control targets.

Ring rot, one of the most destructive diseases of apple worldwide, is caused primarily by Botryosphaeria dothidea and B. kuwatsukai . Here, we sequenced the genomes of B. dothidea strain PG45 (44.3 Mb with 5.12 % repeat rate) and B. kuwatsukai epitype strain PG2 (48.0 Mb with 13.02 % repeat rate), and conducted a comparative analysis of these two genomes, as well as other sequenced fungal genomes, in order to understand speciation and distinctive patterns of evolution of pathogenicity-related genes. Pair-wise genome alignments revealed that the two species are highly syntenic (96.74 % average sequence identity). Both species encode a significant number of pathogenicity-related genes, e.g. carbohydrate active enzymes (CAZYs), plant cell wall degrading enzymes (PCWDEs), secondary metabolites (SMs) biosynthetic enzymes, cytochrome P450 enzymes (CYPs), and secreted peptidases, in comparison to all additional sequenced fungal species involved in various life-styles. The number of pathogenicity-related genes in B. dothidea and B. kuwatsukai is higher than other genomes of Botryosphaeriaceae pathogens ( Macrophomina phaseolina and Neofusicoccum parvum ), suggesting a secondary round of Botryosphaeria -lineage expansion in the family. There were, however, also significant differences in the genomes of the two Botryosphaeria species. Botryosphaeria kuwatsukai , which infects only apple and pear, apparently lost a set of SMs genes, CAZYs and PCWDEs, possibly as a result of host specialization. Botryosphaeria kuwatsukai contained significantly more transposable elements and higher value of repeat induced point (RIP) index than B. dothidea . Our results will be instrumental in understanding how both phytopathogens interact with their plant hosts and in designing efficient strategies for disease control and molecular breeding to help ensure global apple production and food security.

Alternaria spp. are pathogens of several diseases that pose significant threats to apple production. Several putative Alternaria sp. isolates were obtained from lesions of a disease commonly referred to as black dot on apple fruit in Shaanxi Province, China. Pathogenicity tests using mycelial plugs and conidial suspensions indicated that this isolate could cause leaf blotch, as well as moldy core and black dot on fruit. On the basis of sequence analysis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase second largest subunit, and translation elongation factor 1-α, an isolate clustered with the Alternaria sect. Ulocladioides. By combining GAPDH, major allergen Alta1, mating type protein 1-2-1, and the AGA1 gene sequence analysis and morphological description, the isolates were identified as a new species named Alternaria malicola. Our finding expands the documented diversity of apple pathogens within the genus Alternaria and clarifies the taxonomy of the pathogen assemblage that may be associated with three apple diseases.

Sooty blotch and flyspeck (SBFS) fungi are a distinctive group of plant pathogens which, although phylogenetically diverse, occupy an exclusively surface-dwelling niche. They cause economic losses by superficially blemishing the fruit of several tree crops, principally apple, in moist temperate regions worldwide. In this study, we performed genome-wide comparative analyses separately within three pairs of species of ascomycete pathogens; each pair contained an SBFS species as well as a closely related but plant-penetrating parasite (PPP) species. Our results showed that all three of the SBFS pathogens had significantly smaller genome sizes, gene numbers and repeat ratios than their counterpart PPPs. The pathogenicity-related genes encoding MFS transporters, secreted proteins (mainly effectors and peptidases), plant cell wall degrading enzymes, and secondary metabolism enzymes were also drastically reduced in the SBFS fungi compared with their PPP relatives. We hypothesize that the above differences in genome composition are due largely to different levels of acquisition, loss, expansion, and contraction of gene families and emergence of orphan genes. Furthermore, results suggested that horizontal gene transfer may have played a role, although limited, in the divergent evolutionary paths of SBFS pathogens and PPPs; repeat-induced point mutation could have inhibited the propagation of transposable elements and expansion of gene families in the SBFS group, given that this mechanism is stronger in the SBFS fungi than in their PPP relatives. These results substantially broaden understanding of evolutionary mechanisms of adaptation of fungi to the epicuticular niche of plants., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

A warning system for the sooty blotch and flyspeck (SBFS) fungal disease complex of apple, developed originally for use in the southeastern United States, was modified to provide more reliable assessment of SBFS risk in Iowa. Modeling results based on previous research in Iowa and Wisconsin had suggested replacing leaf wetness duration with cumulative hours of relative humidity (RH) ≥97% as the weather input to the SBFS warning system. The purpose of the present study was to evaluate the performance of a RH-based SBFS warning system, and to assess the potential economic benefits for its use in Iowa. The warning system was evaluated in two separate sets of trials-trial 1 during 2010 and 2011, and trial 2 during 2013-2015-using action thresholds based on cumulative hours of RH ≥97% and ≥90%, respectively, in conjunction with two different fungicide regimes. The warning system was compared with a traditional calendar-based system that specified spraying at predetermined intervals of 10 to 14 days. In trial 1, use of the RH ≥97% threshold caused substantial differences between two RH sensors in recording number of hours exceeding the threshold. When both RH thresholds were compared for 2013-2015, on average, RH ≥90% resulted in a 53% reduction in variation of cumulative hours between two identical RH sensors placed adjacent to each other in an apple tree canopy. Although both the SBFS warning system and the calendar-based system resulted in equivalent control of SBFS, the warning system required fewer fungicide sprays than the calendar-based system, with an average of 3.8 sprays per season (min = 2; max = 5) vs. 6.4 sprays per season (min = 5; max = 8), respectively. The two fungicide regimes provided equivalent SBFS control when used in conjunction with the warning system. A partial budget analysis showed that using the SBFS warning system with a threshold of RH ≥90% was cost effective for orchard sizes of >1 ha. The revised warning system has potential to become a valuable decision support tool for Midwest apple growers because it reduces fungicide costs while protecting apples as effectively as a calendar-based spray schedule. The next step toward implementation of the SBFS warning system in the North Central U.S. should be multiyear field testing in commercial orchards throughout the region.

Background: Ectophytic fungi occupy the waxy plant surface, an extreme environment characterized by prolonged desiccation, nutrient limitation, and exposure to solar radiation. The nature of mechanisms that facilitate adaptation to this environment remains unclear. In this study, we sequenced the complete genome of an ectophytic fungus, Ramichloridium luteum, which colonizes the surface of apple fruit, and carried out comparative genomic and transcriptome analysis., Results: The R. luteum genome was 28.18 Mb and encoded 9466 genes containing 1.85% repetitive elements. Compared with cell-penetrating pathogens, genes encoding plant cell wall degrading enzymes (PCWDEs), PTH11-like G protein-coupled receptors (GPCRs) and effectors were drastically reduced. In contrast, genes encoding cutinases and secretory lipases were strikingly expanded, and four of nine secretory lipases were probably acquired by horizontal gene transfer from Basidiomycota. Transcriptomic analysis revealed elevated expression of genes involved in cuticle degradation (cutinase, secretory lipase) and stress responses (melanin biosynthesis, aquaporins, lysozymes and HOG pathway)., Conclusions: Taken together, our results highlight genomic features associated with evolution of surface niche adaptation by the ectophytic fungus R. luteum, namely the contraction of PCWDEs, PTH11-like GPCRs and effectors, and the expansion of cuticle degradation and stress tolerance.

Marssonina blotch, caused by the fungus Marssonina coronariae, is a serious foliar disease on apple in East Asia as well as in other moist temperate regions in Asia, Europe, and South America. Several fungicides were investigated for their toxicity to mycelial growth and conidial germination of the pathogen in vitro. Tebuconazole, kresoxim-methyl, hexaconazole, propiconazole, and a mixture of tebuconazole and benziothiazolinone sharply inhibited mycelial growth but had less effect on conidial germination. Field tests were conducted in a commercial orchard in Baishui County, Shaanxi Province, China, during 2012, 2013, 2014, and 2015 in order to develop recommendations for apple growers. Three applications of tebuconazole, hexaconazole, propiconazole, or a mixture of tebuconazole and benziothiazolinone at 20-day intervals from early July to late August resulted in defoliation incidence of <5%. When sprays of Bordeaux mixture + tebuconazole, Bordeaux mixture + propiconazole, and Bordeaux mixture + tebuconazole and benziothiazolinone were alternated, the spray interval was extended to 25 days and defoliation incidence remained <5%. Based on historical records and our results, scouting for symptoms should begin in mid-June. We recommend commencing the spray period in early July in years with normal rainfall patterns, and spraying in mid- to late June in years with much rainfall. The findings of this study create a foundation for implementation of an efficient spray program against Marssonina leaf blotch in apple orchards in the Loess Plateau Region of China.

Background: The fungal species complex Colletotrichum gloeosporioides sensu lato contains over 20 plant-interacting species. These species exhibit different life styles (e.g., endophytes, foliar and fruit pathogens) and show considerable variation in host and tissue adaptation strategies. Accurate species delimitation in C. gloeosporioides s.l. is very challenging due to nascent lineage boundaries and phenotypic plasticity, which strongly impedes studies of the complex's host-interaction biology. In this study, we first sequenced and compared nine mitogenomes belonging to four C. gloeosporioides s.l. species lineages (C. gloeosporioides, C. fructicola, C. aenigma, and C. siamense s.l.), and evaluated the usefulness of mitogenome sequence in complementing prevailing nuclear markers for species delimitation., Results: The C. gloeosporioides s.l. mitogenomes ranged between 52,671 and 58,666 bp in size, and each contained an identical set of genes transcribed in the same direction. Compared with previously reported Colletotrichum mitogenomes, these mitogenomes were uniquely featured by: (1) significantly larger genome size due to richer intron content and longer intergenic region; (2) striking GC content elevation at the intergenic region; and (3) considerable intron content variation among different species lineages. Compared with nuclear DNA markers commonly used in phylogeny, the mitogenome nucleotide diversity was extremely low, yet the mitogenome alignment contained the highest number of parsimony informative sites, which allowed the generation of a high-resolution phylogeny recognizing all taxonomic lineages, including ones belonging to the very nascent C. siamense s.l. complex. The tree topology was highly congruent with the phylogeny based on nuclear marker concatenation except for lineages within C. siamense s.l. Further comparative phylogenetic analysis indicated that lineage-specific rapid divergence of GS and SOD2 markers confounded concatenation-based species relationship inference., Conclusions: This study sheds light on the evolution of C. gloeosporioides s.l. mitogenomes and demonstrates that mitogenome sequence can complement prevailing nuclear markers in improving species delimitation accuracy. The mitogenome sequences reported will be valuable resources for further genetic studies with C. gloeosporioides s.l. and other Colletotrichum species.

Colletotrichum acutatum, one of the most economically damaging pathogens of strawberry, is the primary causal agent of anthracnose fruit rot (AFR). A key challenge in managing AFR is detecting the pathogen on asymptomatic plants. To meet this need, a loop-mediated isothermal amplification (LAMP) assay was developed that incorporated two sets of primers: LITSG1, targeted on the intergenic transcribed spacer (ITS) region of ribosomal DNA, and Ltub2, on the β-tubulin 2 gene. In pure culture assays, Ltub2 was specific for detection of C. acutatum, whereas LITSG1 detected C. acutatum and two additional anthracnose pathogens, C. gloeosporioides and C. fragariae. LITSG1 had 10-fold lower detection threshold (20 pg of mycelial DNA) than Ltub2 (200 pg mycelial DNA) in detection of C. acutatum from pure culture. For detection on asymptomatic leaves, two protocols for dislodging C. acutatum for DNA extraction were compared: i) the sonicate-agitate (SA) method and ii) the freeze-incubate-sonicate-agitate (FISA) method, which initially freezes tissues, followed by 2 days of incubation at 26°C in darkness, and then, sonication and agitation. Both methods were used for greenhouse-grown plant leaves that had been spray inoculated with serial dilutions ranging from 1.5 × 10 6 to 1.5 conidia ml -1 . The FISA method produced more repeatable results than the SA method. For the FISA method, detection limits (expressed as initial inoculum concentrations) using LITSG1 and Ltub2 were 1.5 × 10 1 and 1.5 × 10 2 conidia ml -1 , respectively. For composite samples comprised of inoculated (1.5 × 10 6 conidia ml -1 ) and noninoculated leaves of greenhouse-grown strawberry, the two sets of LAMP primers were compared using the SA method. Primer set LITSG1 consistently detected the pathogen from a single inoculated leaf in bulk samples of 50 or fewer pathogen-free leaves, whereas Ltub2 consistently detected one inoculated leaf in 20 or fewer pathogen-free leaves. Using primer set LITSG1, FISA was more sensitive than SA for detecting C. acutatum on leaves of field-grown plants from Florida. In an Iowa field trial using the FISA method, both primer sets detected C. acutatum in samples of asymptomatic leaves 6 days before fruit symptoms appeared. The results indicate that the LAMP assay has potential to provide a simplified method for detection of C. acutatum on asymptomatic strawberry plants.

Valsa canker, caused by the fungus Valsa mali, is one of the most destructive diseases of apple in the primary production areas of China and other East Asian countries. Currently, there are no effective control methods for this disease. We investigated the occurrence of Valsa canker in 24 apple orchards in Shaanxi Province in concert with foliar nutrient analysis, and found that there was a significant negative correlation of leaf potassium (K) content with incidence and severity of Valsa canker. Fertilization experiments showed that increasing tree K content enhanced resistance to pathogen colonization and establishment. Apple trees with leaf K content greater than 1.30% exhibited almost complete resistance to Valsa mali. Field trials demonstrated that increasing K fertilization could significantly reduce disease incidence. Improved management of tree nutrition, especially K content, could effectively control the occurrence and development of Valsa canker.

Modern industrial agriculture depends on high-density cultivation of genetically similar crop plants, creating favorable conditions for the emergence of novel pathogens with increased fitness in managed compared with ecologically intact settings. Here, we present the genome sequence of six strains of the cucurbit bacterial wilt pathogen Erwinia tracheiphila (Enterobacteriaceae) isolated from infected squash plants in New York, Pennsylvania, Kentucky, and Michigan. These genomes exhibit a high proportion of recent horizontal gene acquisitions, invasion and remarkable amplification of mobile genetic elements, and pseudogenization of approximately 20% of the coding sequences. These genome attributes indicate that E. tracheiphila recently emerged as a host-restricted pathogen. Furthermore, chromosomal rearrangements associated with phage and transposable element proliferation contribute to substantial differences in gene content and genetic architecture between the six E. tracheiphila strains and other Erwinia species. Together, these data lead us to hypothesize that E. tracheiphila has undergone recent evolution through both genome decay (pseudogenization) and genome expansion (horizontal gene transfer and mobile element amplification). Despite evidence of dramatic genomic changes, the six strains are genetically monomorphic, suggesting a recent population bottleneck and emergence into E. tracheiphila's current ecological niche., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Sooty blotch and flyspeck (SBFS) fungi are unconventional plant pathogens that cause economic losses by blemishing the surface appearance of infected fruit. Here, we introduce the 18.14-Mb genome of Peltaster fructicola, one of the most prevalent SBFS species on apple. This undersized assembly contains only 8,334 predicted protein-coding genes and a very small repertoire of repetitive elements. Phylogenomics and comparative genomics revealed that P. fructicola had undergone a reductive evolution, during which the numbers of orphan genes and genes involved in plant cell wall degradation, secondary metabolism, and secreted peptidases and effectors were drastically reduced. In contrast, the genes controlling 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis and appressorium-mediated penetration were retained substantially. Additionally, microscopic examination of the surfaces of infected apple indicated for the first time that P. fructicola can not only dissolve epicuticular waxes but also partially penetrate the cuticle proper. Our findings indicate that genome contraction, characterized mainly by the massive loss of pathogenicity-related genes, has played an important role in the evolution of P. fructicola (and by implication other SBFS species) from a plant-penetrating ancestor to a non-invasive ectophyte, displaying a novel form of trophic interaction between plants and fungi.

Members of the sooty blotch and flyspeck (SBFS) complex are epiphytic fungi in the Ascomycota that cause economically damaging blemishes of apples worldwide. SBFS fungi are polyphyletic, but approx. 96% of SBFS species are in the Capnodiales. Evolutionary origins of SBFS fungi remain unclear, so we attempted to infer their origins by means of ancestral state reconstruction on a phylogenetic tree built utilizing genes for the nuc 28S rDNA (approx. 830 bp from near the 59 end) and the second largest subunit of RNA polymerase II (RPB2). The analyzed taxa included the well-known genera of SBFS as well as non-SBFS fungi from seven families within the Capnodiales. The non-SBFS taxa were selected based on their distinct ecological niches, including plant-parasitic and saprophytic species. The phylogenetic analyses revealed that most SBFS species in the Capnodiales are closely related to plant-parasitic fungi. Ancestral state reconstruction provided strong evidence that plant-parasitic fungi were the ancestors of the major SBFS lineages. Knowledge gained from this study may help to better understand the ecology and evolution of epiphytic fungi., (© 2016 by The Mycological Society of America.)

Sooty blotch and flyspeck (SBFS) is a fungal disease complex that can cause significant economic losses to apple growers by blemishing the fruit surface with dark-colored colonies. Little is known about the phenology of host infection for this diverse group of epiphytes. In 2009 and 2010, we investigated the timing of infection of apple fruit by SBFS species in six commercial apple orchards in Iowa. Five trees in each orchard received no fungicide sprays after fruit set. Within 3 weeks after fruit set, 60 apples per tree were covered with Japanese fruit bags to minimize inoculum deposition. Subsequently, a subsample of bagged apples was exposed for a single 2-week-long period and then rebagged for the remainder of the growing season. Experimental treatments included seven consecutive 2-week-long exposure periods; control treatments were apples that were either bagged or exposed for the entire season. After apples had been stored at 2°C for 6 weeks following harvest, all SBFS colonies on the apples were identified to species using a PCR-RFLP protocol. A total of 15 species were identified. For the seven most prevalent species, the number of infections per cm 2 of fruit surface was greatest on apples that had been exposed early in the season. Two SBFS species, Peltaster fructicola and Colletogloeopsis-like FG2, differed significantly from each other in time required to attain 50% of the total number of colonies per apple, and analysis of variance indicated a significant interaction of SBFS taxon with exposure period. Our findings are the first evidence of species-specific patterns in timing of SBFS inoculum deposition and infection on apple fruit, and strengthen previous observations that most SBFS infections resulting in visible colonies at harvest develop from infections that occur early in the fruit development period. By defining taxon-specific phenological patterns of fruit infection, our findings, when combined with knowledge of region-specific patterns of taxon prevalence, provide a foundation for development of more efficient and cost-effective SBFS management tactics.

The genus Cyphellophora includes human- and plant-related species from mammal skin and nails, plant materials, and food. On the basis of analysis of ITS, LSU, TUB2 and RPB1 data and morphological characters, three new species, Cyphellophora phyllostachysdis, C. artocarpi and C. musae, associated with sooty blotch and flyspeck disease, were added to this genus. The 2D structure of ITS1 and ITS2 confirmed this taxonomic status. Pathogenicity tests on apple fruit indicated that C. artocarpi could be a sooty blotch and flyspeck pathogen of apple.

Bacterial wilt threatens cucurbit crop production in the Midwestern and Northeastern United States. The pathogen, Erwinia tracheiphila, is a xylem-limited bacterium that affects most commercially important cucurbit species, including muskmelon, cucumber, and squash. Bacterial wilt is transmitted and overwintered by striped and spotted cucumber beetles. Since there are few commercially available resistant cultivars, disease management usually relies on use of insecticides to suppress vector populations. Although bacterial wilt was initially described more than 100 years ago, our knowledge of disease ecology and epidemiology advanced slowly for most of the 20th century. However, a recent wave of research has begun to fill in missing pieces of the bacterial wilt puzzle. This article-the first review of research toward understanding the cucurbit bacterial wilt pathosystem-recounts early findings and updates our understanding of the disease cycle, including pathogen and vector biology. We also highlight research areas that could lead to more efficient and ecologically based management of bacterial wilt.