Changes in planta K nutrient content altered the interaction pattern between Nicotiana benthamiana and Alternaria longipes.

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.

First report of cucurbit yellow vine disease caused by Serratia marcescens on cucurbit crops in Iowa.

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 (~108 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.

Cloning of maize chitinase 1 gene and its expression in genetically transformed rice to confer resistance against rice blast caused by Pyricularia oryzae.

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.

CfCpmd1 Regulates Pathogenicity and Sexual Development of Plus and Minus Strains in Colletotrichum fructicola Causing Glomerella Leaf Spot on Apple in China.

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.

Sufficient coumarin accumulation improves apple resistance to Cytospora mali under high-potassium status.

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.

Guiding STEM graduate students to better speaking skills.

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.

First report of Fusarium sporotrichioides causing Fusarium fruit rot on peaches in Pakistan.

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 ß-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 ß-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 ß-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.

Diversity of Colletotrichum Species Causing Apple Bitter Rot and Glomerella Leaf Spot in China.

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 TUB2) 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.

The CfMcm1 Regulates Pathogenicity, Conidium Germination, and Sexual Development in Colletotrichum fructicola.

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 ΔCfMcm1 lost pathogenicity to apple leaves by inhibiting conidial germination and appressorium formation. In addition to appressorium-mediated pathogenicity, ΔCfMcm1 colonization and hyphal extension in wounded apple fruit was also reduced, and conidial germination mode and conidial color were altered. ΔCfMcm1 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 ΔCfMcm1 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.

Expression and Functional Analysis of the Type III Secretion System Effector Repertoire of the Xylem Pathogen Erwinia tracheiphila on Cucurbits.

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.

First Report of Colletotrichum scovillei Causing Anthracnose on Watermelon (Citrullus lanatus) in Malaysia.

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.

Sustainable Apple Disease Management in China: Challenges and Future Directions for a Transforming Industry.

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.

The Contributions to Virulence of the Effectors Eop1 and DspE Differ Between Two Clades of Erwinia tracheiphila Strains.

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.

Comparison of mitochondrial genomes provides insights into intron dynamics and evolution in Botryosphaeria dothidea and B. kuwatsukai.

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.

First Report of a Ceratocystis manginecans Causing Quick Decline of Psidium guajava in Pakistan.

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.

Transcription Factor CfSte12 of Colletotrichum fructicola Is a Key Regulator of Early Apple Glomerella Leaf Spot Pathogenesis.

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 (ΔCfSte12) 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 ΔCfSte12 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.

Sequence analysis of 16S rDNA, gyrB and alkB genes of plant-associated Rhodococcus species from Tunisia.

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, gyrB and alkB genes was used to compare our strains with type strains of Rhodococcus. For most of our strains, sequence similarity of the 16S rDNA, gyrB and alkB 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 gyrB sequences provided a more precise classification of our strains to species level than 16S rDNA sequences, whereas analysis of alkB sequences was unable to identify strains with orange-coloured colonies to species level.

Performance and Profitability of Rain-Based Thresholds for Timing Fungicide Applications in Soybean Rust Control.

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.

Precipitation Impacts Dissemination of Three Sooty Blotch and Flyspeck Taxa on Apple Fruit.

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.