Meta-analysis in Plant Pathology: Publication Patterns, Methodological Trends and Potential Future Directions.

Meta-analysis (MA) is increasingly adopted in agricultural and ecological sciences, fields at the interface with plant pathology. Our review on the use of MA in the field, since 1999, identified 79 original research papers. Fifty percent of these works were published in two APS journals, emphasizing their central role in disseminating MA research. Analysis of the number of authors per study and the authorship network revealed a tightly knit field. The network shows a few "hub" authors or groups that significantly influence research directions, with a clear geographical clustering in North and South America. Most of the MA papers focused on the effects of disease control, where fungicide was the main product. The MA investigations often focus on response variables like yield and disease severity for which the analyzed effect sizes differ, but the log of the ratio of means and untransformed means are predominant. The study selection to be included in the MA often follows systematic review standards or when clear quantitative criteria are used for study inclusion. Yet, the inclusion of data from reports, newsletters, and research trial data adds breadth. Frequentist rather than Bayesian inference is most common and SAS and R are the preferred software. Our review underscores the escalating importance of MA in plant pathology, especially for determining the mean and variability of the effects of different control methods on disease and yield. Efforts should continue to improve the detailed reporting of MA methods used, data sharing and reporting of measures of heterogeneity among studies.

First Report of Tomato Yellow Mottle-Associated Virus Infecting Tobacco in China.

Tomato yellow mottle-associated virus (TYMaV) belongs to the genus Cytorhabdovirus in the family Rhabdoviridae and has been reported to infect a variety of Solanaceae crops, such as Solanum lycopersicum, S. nigrum, Capsicum annuum and Nicotiana benthamiana (Li et al. 2022, Li et al. 2023, Xu et al. 2017, Zhou et al. 2019). In August 2022, about 500 out of 2000 tobacco (N. tabacum) plants showing leaf distortion, crinkling and mosaic symptoms were found in one tobacco growing field in Xingren City, Guizhou Province, China. To identify the causal pathogen(s), leaves from 20 symptomatic tobacco plants were collected and pooled to perform small RNA deep sequencing (sRNA-Seq) and assembly. Briefly, total RNA was extracted with TRIzol Reagent (Takara, Kusatsu, Japan). A small RNA cDNA library was constructed by the small RNA Sample Pre Kit. sRNA-Seq was performed with an Illumina NovaSeq 6000 platform. About 29 million reads were obtained and 334 contigs generated after removal of host-derived sequences. Among them, 31 unique contigs mapped to the TYMaV genome (NC_034240.1), covering 28.43% of the genome with the mean read coverage of 0.92%. Meanwhile, 226 contigs mapped to the genome of a potyvirus, chilli veinal mottle virus (ChiVMV, NC_005778.1), covering 88.79% of the genome with the mean read coverage of 0.83%. To verify the sRNA-Seq result for TYMaV identification, reverse transcription (RT)- PCR was performed with specific primers TYMaV-F (5'-CTGACGTAGTGTTGGCAGAT-3') and TYMaV-R (5'-AACCTCCATGCAGAACCATGG-3'). The expected-size 936-bp fragment was amplified from total RNA of all 20 samples. Dot enzyme-linked immunosorbent assays (Dot-ELISA) with antibody for TYMaV (kindly provided by Dr. Zhenggang Li from Guangdong Academy of Agricultural Sciences) were performed and further verified TYMaV infection. In addition, five asymptomatic tobacco plants from the same field as controls were used to detect TYMaV by RT-PCR and Dot-ELISA, and all samples showed negative test results. Subsequently, 17 primer pairs (Supplementary Table 1) were used to obtain the full-length sequence of TYMaV from a single positive tobacco sample by RT-PCR, followed by Sanger sequencing at Sangon Biotech (Shanghai, China). The resulting amplicon sequences were assembled into a nearly full-length genome sequence of a TYMaV isolate from tobacco in Guizhou (TYMaV-GZ). BLASTn analysis of the 13, 393 nt-long sequence (GeneBank accession number, PP444718) revealed 84.7% and 87.2% nt sequence identity with the TYMaV tomato isolate (KY075646.1) and the TYMaV S. nigrum isolate (MW527091.1), respectively. Moreover, five S. nigrum plants showing leaf crinkling and mosaic symptoms from tobacco fields tested positive for TYMaV by RT-PCR assay, suggesting a potential spread of TYMaV between tobacco and S. nigrum, which may serve as a reservoir for the virus in the tobacco fields. However, the transmission route of TYMaV remains unknown, and further verification is needed. To our knowledge, this is the first report of TYMaV infecting tobacco crop in China. It will be important to assess the potential economic importance of TYMaV to tobacco production in China and elsewhere, and to elucidate the respective roles of this virus and ChiVMV in the leaf distorting and yellowing symptoms.

Pathological characterization and management of Lasiodiplodia theobromae, a hemi-biotroph with an interkingdom host range.

Heart rot disease, caused by Lasiodiplodia theobromae, is destructive for date palms and other woody plants. The disease was reported in several oasis in Egypt, and the pathogen was found in association with infected trees suffering die-back and rachis blight. Seven phylogenetically distinct fungal isolates were selected, and their pathogenicity was confirmed on date palms. The isolates exhibited variable degrees of virulence on inoculated leaves, which confirms the variation. We examined the antifungal effect of microbial bioagents and plant extracts on heart rot disease. The isolates of Trichoderma spp. gave moderate reduction of the pathogen's linear growth (40-60%), while their exudates were ultimately ineffective. Bacillus spp. isolates, except for B. megaterium, were more effective against spore germination as they gave 80-90% reduction on average. Among the examined plant extracts garlic sap gave 98.67% reduction of linear growth followed by artemisia (15.5%) and camphor (24.8%). The extraction methods greatly influenced the antifungal efficiency of each extract as exposure to organic solvents significantly decreased the efficiency of all extracts, while hot water extraction negatively affected garlic sap only. Successful bioagents and plant extracts were further assayed for the suppression of heart rot disease on date palms. Both T. album and T. harzianum gave comparable degrees of suppression as by commercial fungicides. In addition, treatment before or during pathogen inoculation was the most effective as it significantly enhanced the expression of defense-related enzymes. Our findings suggest bio-pesticides possessing a dual role in disease suppression and defense boosters for date palms suffering heart rot disease.

Development of a TaqMan assay for the detection of 'Candidatus Phytoplasma brasiliense' and assessment by high resolution melt-curve analysis (HRMA).

'Candidatus Phytoplasma brasiliense' (CPB) is a phytoplasma originally discovered in South America and is known to infect a wide variety of economically important crops. It is most prevalent in Hibiscus spp. where it causes witches broom symptoms and papaya where it causes bunchy top. Recently, CPB was documented for the first time in North America in a new host, globe sedge. In this study two qPCR assays are developed, one utilizing high resolution melt curve analysis (HRMA) based on the secA gene and the other a TaqMan assay based on the dnaK gene. The secA/HRMA and dnaK/TaqMana ssay successfully amplified isolates of CPB. Both assays were screened against available isolates of 16SrI, 16SrII and 16SrIV phytoplasmas. The secA/HRMA assay failed to amplify 16SrI, 16SrIII and 16SrIV phytoplasmas but successfully amplified 16SrII phytoplasmas. The resulting Tm products of CPB and 16SrII phytoplasmas displayed a difference of 0.5°C difference, easily distinguishing them by melt curves. The dnaK/TaqMan assay failed to amplify all non-CPB phytoplasma isolates in the study. The development of these assays provides a valuable tool that will significantly improve monitoring programs in Florida and will aid in developing a better fundamental understanding of the epidemiology of this phytoplasma.

Arthrocladiella mougeotii causing powdery mildew on goji berry plants (Lycium barbarum and L. chinense) and mixed infections with Phyllactinia chubutiana in California.

Goji berries (Lycium barbarum and L. chinense) have a rich historical significance in traditional Chinese medicine and have gained popularity as a superfood in Western cultures. From 2021 to 2023, powdery mildew was observed on goji plants of both species in community and residential gardens in Yolo County, California (USA). Disease severity varied from 20 to 100% of infected leaves per plant. Powdery mildew was characterized by the presence of white fungal colonies on both sides of leaves and fruit sepals. Additionally, a brownish discoloration was observed in infected mature leaves, resulting in further defoliation. Morphologically, the fungus matched the description of Arthrocladiella mougeotii. The pathogen identity was confirmed by phylogenetic analyses of the rDNA internal transcribed spacer and the 28S rDNA gene sequences. Pathogenicity was confirmed by inoculating healthy L. barbarum plants using infected leaves and successfully reproducing powdery mildew symptoms after 28 days (22°C, 60% RH), with A. mougeotii colonies confirmed by morphology. Control leaves remained symptomless. Co-infection with Phyllactinia chubutiana was detected on plants from two separate gardens, with A. mougeotii observed first in late spring (May to June) and P. chubutiana later in the summer (July to August). These results revealed that both A. mougeotii and P. chubutiana constitute causal agents of powdery mildew on goji berry plants, often infecting the same plant tissues simultaneously. To our knowledge, this is the first report of A. mougeotii causing powdery mildew on L. barbarum and L. chinense in California, which provides a better understanding of the etiology of powdery mildew of goji plants in California.

First Report of Exserohilum rostratum Causing Brown Spot on Spartina alterniflora in China.

Spartina alterniflora Loisel, a perennial grass, has become an invasive species in China's coastal wetlands (Zhang et al. 2018). In July 2021, brown spot symptoms were observed on S. alterniflora in a coastal wetland (21°45'48″N, 108°44'00″E) in Beihai City, Guangxi Province, China. The disease affected approximately 50% of the plants in the surveyed area (0.2 ha) and was also observed in other regions of Beihai. It caused brown lesions with a gray or whitish center on the leaves and stems of S. alterniflora. As the disease developed, it ultimately led to leaf shedding and plant death. To isolate the causal agent, 18 fragments (~ 5 mm) from six symptomatic plants (3 leaf pieces per plant) were surface sterilized with 1% NaOCl solution for 2 min and rinsed three times with sterilized water. Subsequently, the tissues were placed on potato dextrose agar (PDA) medium supplemented with chloramphenicol (0.1 g/liter) and incubated at 28°C for three days. The hyphal tips were transferred onto fresh PDA to obtain pure cultures. A total of 25 isolates were obtained, 20 of which shared similar morphologies, while the remaining five exhibited distinct morphological characteristics and were non-pathogenic to S. alterniflora. Three isolates (MC16.1.3, MC16.6.2, and MC16.8.3) were randomly selected from the 20 for further investigation. The colonies on PDA were flat with dense aerial mycelia. The colony margins were entire, light brown in the centre, white to grey at the margin; reverse dark brown in the centre, gray at the margin. Conidia were straight to slightly curved, light olive-brown to dark olive-brown, septate, measured 33.5 to 79.1 µm × 10.4 to 18.7 µm (average 52.9 × 14.4 µm, n = 100), with a distinctly protruding hilum swelled from the basal cell. For molecular identification, the genomic DNA was extracted from mycelium on PDA using the CTAB method (Guo et al. 2000). The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor 1 alpha (TEF1-α) genes were amplified and sequenced with the primer pairs ITS1/ITS4 (White et al. 1990), GPD1/GPD2 (Berbee et al. 1999), and EF1-983/EF1-2218 (Rehner et al. 2005), respectively. A BLAST analysis revealed that the ITS (OR516787-9), GAPDH (OR523686-8), and TEF-α (OR523683-5) had 99.1 to 99.7% identity with those of E. rostratum strains BRIP 11417 (LT837836, LT882553, and LT896656) and CBS 128061 (KT265240, LT715900, and LT896658) (Hernández-Restrepo et al. 2018). Based on the concatenated sequences, a phylogenetic tree generated by PhyloSuite software (Zhang et al., 2020) through Bayesian inference (BI) and Maximum Likelihood (ML) methods placed the isolates within E. rostratum. These morphological characteristics and molecular analyses confirmed the pathogen as E. rostratum (Hernández-Restrepo et al. 2018; Kaboré et al. 2022). To confirm pathogenicity, a conidial water suspension (~ 1 × 106 conidia/ml) of each of the three strains was inoculated on nine healthy S. alterniflora plants that had been grown for six months. Control plants were treated with sterile water. All plants were then enclosed in plastic bags and incubated in a greenhouse at 28°C. Six days after inoculation, the plants exhibited symptoms similar to those observed in nature. The control plants developed no symptoms. These experiments were replicated three times with similar results. To fulfill Koch's postulates, E. rostratum was consistently re-isolated from symptomatic tissue and confirmed by morphology and sequencing, whereas no fungus was isolated from the control plants. In recent years, S. alterniflora has posed a serious threat to the indigenous biodiversity of wetland ecosystems (Zhang et al. 2018). To our knowledge, this is the first report of E. rostratum causing brown spot on S. alterniflora worldwide.

Detection of Podosphaera macularis in Air Samples by Quantitative PCR.

Detection and quantification of pathogen propagules in the air or other environmental samples is facilitated by culture-independent assays. We developed a quantitative PCR assay for the hop powdery mildew fungus, Podosphaera macularis, for detection of the organism from air samples. The assay utilizes primers and a TaqMan probe designed to target species-specific sequences in the 28S large subunit (LSU) of the nuclear ribosomal rDNA. Analytical sensitivity was not affected by the presence of an exogenous internal control or potential PCR inhibitors associated with DNA extracted from soil. The level of quantification of the assay was between 200 and 350 conidia when DNA was extracted from a fixed number of conidia. The assay amplified all isolates of P. macularis tested and had minimal cross-reactivity with other Podosphaera species when assayed with biologically relevant quantities of DNA. Standard curves generated independently in two other laboratories indicated that assay sensitivity was qualitatively similar and reproducible. All laboratories successfully detected eight unknown isolates of P. macularis and correctly discriminated Pseudoperonospora humuli and a water control. The usefulness of the assay for air sampling for late-season inoculum of P. macularis was demonstrated in field studies in 2019 and 2020. In both years, airborne populations of P. macularis in hop yards were detected consistently and increased during bloom and cone development.

First Report of Kiwifruit Root Rot Caused by Globisporangium spinosum in China.

Kiwifruit is widely cultivated for its high vitamin C content and nutritional value. In January 2022, root rot symptoms were found in about 30% of Actinidia chinensis cv. Jinyan plants grafted on A. deliciosa rootstocks in an orchard located in Sanming (26.32°N, 117.23°E), Fujian Province of China. The affected plants appeared stunted, with brown and decaying roots, some of which were covered with white hyphae. To isolate the pathogen, the surfaces of typical symptomatic roots were sterilized for 30 s using 75% ethanol, followed by four rinses in sterile water, placing on potato dextrose agar (PDA), and incubating away from light at 25°C for 7 days. 16 Globisporangium-like isolates were obtained through hyphal tip isolation, displaying a milky-white appearance with irregular protuberances on the surface, and yellow-white backs with radial fold lines. The isolates were then cultured on corn meal agar for 5 days at 25°C in dark for morphological characteristics. Under microscope, the hyphae appeared as long strips without septa and 4.1 to 8.2 µm wide (average 6.7 µm), containing irregularly sized spherical droplets. Both terminal and intercalary hyphae swellings were observed; these appeared either spherical or subspherical, with some having projections. Their dimensions were 12.3 to 27.6 µm (average 17.3 µm). The oospores were mostly spherical, either plerotic or aplerotic, 11.8 to 22.3 µm wide (average 18.9 µm), with occasional projections. The antheridia were rod-shaped and curved, with one end attached to the oogonia. Amplification of the sequences of internal transcribed spacer (ITS) regions and cytochrome c oxidase subunit I (COI) were conducted using the primers ITS1/ITS4 (White et al. 1990) and OomCoxI-Levlo/OomCoxI-Levup (Robideau et al. 2011), respectively. The sequencing results revealed identical ITS and COI sequences in all 16 isolates. BLASTn analysis of the 969-bp ITS sequence ON202808 showed 99.38-99.59% similarity (965/971bp, 967/971bp) with the KJ162353 and AY598701 sequences from Globisporangium spinosum isolates, while the 700-bp COI sequence ON075783 showed 100% and 99.41% identity (680/680bp, 676/680bp) with the GenBank sequences HQ708835 and HQ708832, respectively, from G. spinosum. Phylogenetic analysis also showed that the obtained isolate (termed MA16) clustered with isolates from G. spinosum on the same evolutionary branch. For pathogenicity testing, four-month-old healthy Jinyan (A. chinensis) plants grown in sterilized media were transferred to sterile petri dishes covered with wet filter paper, and their roots were inoculated with a 5-mm-wide disk of MA16 when cultivated on PDA medium for 5 days. Miliang-1 (A. deliciosa) and Hongyang (A. chinensis) plants were treated similarly. The control groups each included three plants that were inoculated with non-colonized PDA. The plants were kept at 25 °C with a 12-/12-h light/dark cycle for 10 days when the inoculated plants exhibited root rot symptoms similar to those seen in the field, together with rotting and browning of the leaves. The control plants appeared healthy with no symptoms. After re-isolated from infected tissues, the pathogen was verified to be G. spinosum according to its ITS sequence, thus fulfilling the Koch's postulates. Recently, Pythium spinosum has been classified as G. spinosum according to whole-genome sequencing and phylogenomic analysis (Nguyen et al. 2022). Based on the morphological features and pathogenicity results, MA16 was identified as G. spinosum (van der Plaats-Niterink 1981; Huo et al. 2023). This report appears to be the first description of kiwifruit root rots caused by G. spinosum in China, and its identification will assist the development of strategies to counteract the disease.

Construction of an infectious clone of citrus chlorotic dwarf-associated virus and confirmation of its pathogenicity.

Citrus chlorotic dwarf disease (CCDD) seriously affects the citrus industry. Citrus chlorotic dwarf-associated virus (CCDaV) is speculated to be the causal agent of CCDD. However, this speculation has not been confirmed by fulfilling Koch's postulates. In this study, an infectious clone was constructed that comprises a 1.6-fold tandem CCDaV genome in the binary vector pBinPLUS and agro-inoculated into Eureka lemon (Citrus limon) seedlings through vacuum infiltration. At 60 days post inoculation, 25% of the Eureka lemon seedlings developed symptoms of crinkling and curling that are the same as those associated with the wild-type virus. Western blotting and graft transmission assays confirmed that the infectious clone systemically infected Eureka lemon seedlings. In addition, CCDaV can establish infection on three more Citrus species and one hybrid, although at different infection rates. These findings support that CCDaV is the primary causal agent of CCDD. The infectious CCDaV clone will allow further studies on the functions of viral proteins and molecular interactions of CCDaV with its hosts.

First Report of Anthracnose Caused by Colletotrichum siamense on Machilus thunbergii Siebold & Zucc. in South Korea.

Machilus thunbergii Siebold & Zucc., known as Japanese bay tree, is an evergreen tree distributed widely in East Asia, including South Korea, where the species is of ecological importance. Machilus thunbergii provides habitat for wildlife species and is a common urban tree. In September 2022, anthracnose symptoms on leaves were observed in Jeju (33°26'02.4"N, 126°19'48.8"E) and Tongyeong (34°49'27.1"N, 128°24'01.8"E) in South Korea. Disease incidence on leaves of each affected tree, naturally growing in an urban forest area covering approximately 0.5 ha was approximately ~ 70 % in each study area. Anthracnose symptoms that were observed on 70 to 80% leaves per tree in each study area included orbicular or irregular, whitish-grey spots on leaves that were 1.5 to 3.0 cm in diam. In some cases where leaves were severely affected, larger blotches were formed, leading to bleaching symptoms and eventually defoliation. For pathogen isolation, two or three leaves showing anthracnose symptoms from each of the 15 trees were randomly selected and brought to the laboratory. Fungal isolations were then directly made by transferring spores from acervuli that developed on diseased leaves onto potato dextrose agar (PDA) media. Cushion shaped acervuli filled with salmon to orange-colored conidial masses were produced on media approximately two weeks after the incubation at 25 ± 1°C with a photoperiod of 12 h. Conidia were single celled, hyaline, cylindrical with rounded ends, smooth walls, 13.7 to 18.1 µm long and 3.1 to 4.5 µm wide (n=30). Among 15 cultures that were successfully isolated, 10 isolates were retained based on culture characteristics, and two randomly selected monoconidial cultures were deposited in the culture collection (CDH) of the Chungnam National University, Republic of Korea (Accession No. CDH057-58). Two isolates selected, CDH057 and CDH058, were subjected to identification, and this was achieved based on multiplesequence comparisons using on internal transcribed spacer regions of rDNA (ITS1 and ITS2), partial sequences of actin (ACT) and ß-tubulin (TUB2) gene regions amplified using ITS1F / ITS4, ACT-512F / ACT-783R and T1 / Bt2b, respectively (Weir et al. 2012). The representative sequence data were deposited in GenBank under the accession numbers OR473277 and OR473278 for the ITS, OR480772 and OR480773 for ACT, and OR480774 and OR480775 for TUB2. The resulting sequences were further used for a phylogenetic analysis based on the maximum likelihood method using a concatenated dataset of the ITS, ACT and TUB2 gene sequences for Colletotrichum species in the C. gloeosporioides clade. The results showed that the pathogen isolated in this study clustered with Colletotrichum siamense (Vouchered specimens: MFLU 090230, COUFPI291, and COUFPI294) (Prihastuti et al. 2009). Sequence comparisons revealed that the isolates obtained in this study differed from the type species of C. siamense (MFLU 090230; FJ972613 for ITS, FJ 907423 for ACT, FJ907438 for TUB2) at 2 of 258 bp (∼0.8%) and 6 of 387 bp (∼1.6%) in the ACT and TUB2 sequences, respectively, while the ITS was identical to the type species. For pathogenicity tests, a total of ten three-year-old seedlings of M. thunbergii were used. The leaves of each tree were sprayed with 5 ml of conidial suspension (105 conidia/ml, isolate CDH057). Three control plants were sprayed with sterile water. After being sprayed, treated areas were sealed with a plastic bag for 24 hours to preserve humidity. Anthracnose symptoms, identical to those observed in the field, appeared five to seven days after the inoculations, while no symptoms were observed on control plants. The isolates used in the pathogenicity test were reisolated from 90% of lesions, and their identity was confirmed based on sequence comparisons, thus fulfilling Koch's postulates. Species of the C. gloeosporioides species complex include important plant pathogens, particularly C. siamense, which cause significant losses of economic and ecological relevance on a wide range of hosts (~ 100 hosts) (Talhinhas and Baroncelli 2021). Although C. fioriniae in the C. acutatum species complex, was found on M. thunbergii in South Korea (Thao et al. 2023), anthracnose associated with C. siamense on M. thunbergii has not been reported in the country. In this regard, this is the first report of anthracnose caused by C. siamense on M. thunbergii in South Korea. To effectively control the disease, more attention should be paid on the host range of the pathogen and other regions where the disease caused by the pathogen might occur in the country.

Molecular-Phylogenetic Characterization of Xanthomonas hortorum pv. pelargonii Strains Causing Leaf Spot of Geraniums in Iran.

Bacterial blight and leaf spot of geraniums is a destructive disease of cultivated Pelargonium species around the world. During 2020-2021, surveys were conducted in seven geranium-growing provinces of Iran to monitor the status of bacterial blight and leaf spot disease. The disease was observed in six surveyed provinces varying in the extent of occurrence and severity. Twenty-two Gram-negative pale-yellow bacterial strains resembling members of Xanthomonas were isolated from symptomatic leaves and stems. Pathogenicity and host range assays showed that the bacterial strains were pathogenic on Pelargonium grandiflorum, P. graveolens, P. peltatum, and P. zonale. All strains were positive for PCR test using the primer pair XcpM1/XcpM2 which is specific for Xanthomonas hortorum pv. pelargonii. Phylogenetic analysis using the sequences of gyrB and lepA genes showed that the 22 strains clustered in a clade among the sequences of X. hortorum pv. pelargonii strains retrieved from the GenBank, while distinct from the other pathovars of X. hortorum. BOX-PCR-based fingerprinting using BOX-A1R primer revealed that the strains isolated in this study were grouped into two clusters while no distinct correlation was observed between the host/area of isolation and BOX-PCR fingerprinting. None of the strains obtained in this study nor reference strain of the pathogen did produce bacteriocin against each other. Results obtained in this study shed light on the geographic distribution, taxonomic status and host range of the bacterial blight and leaf spot pathogen of geraniums in Iran, paving the path of further research on disease management.

First Report of Charcoal Rot on Tobacco Caused by Macrophomina phaseolina in Hunan Province, China.

Flue-cured tobacco (Nicotiana tabacum L.) is a significant cash crop globally. In August 2022, necrotic lesions on stem associated with root rot and wilting were observed on flue-cured tobacco (Cv. Yunyan 87) in fields located in Banxin village (27.95N,109.60E) of Fenghuang county in Xiangxi Autonomous Prefecture, Hunan Province, China. The affected and damaged area of tobacco is approximately 10 hectares, with adisease incidence of 60%. Lots of small black speckling within the lower stem of the affected plant, vascular tissue changed to black, dry rot, and looked like charcoal breezes. Small pieces were cut from healthy and diseased tissues, surface sterilized with 5% NaClO for 3 min and 75% ethanol for 1 min, rinsed with sterile distilled water and air-drying, incubated on oat medium incubated at 28℃ for five days. These isolates grew fast and produced typical black microsclerotia. The morphological were septate hyphae and microsclerotia. The microsclerotia were black and regularly round, with a 42.5 - 92.9 µm diameter. These morphological features were consistent with Macrophomina phaseolina (Smith and Wyllie 1999). The internal transcribed spacer (ITS) rDNA and translation elongation factor 1-α (TEF1-α) genes of three representative isolates were amplified and sequenced using the primers ITS1/ITS4 and EF1-728F/EF2R (Machado et al. 2019). Our resulting sequences (GenBank accessions OR435093, OR435101, OR435102 for ITS; OR891780, OR891781 and OR891782 for EF1-α) showed 99-100% similarity with M. phaseolina by NCBI blast. Phylogenetic analysis was conducted using MEGA-X software with the NJ method. The combined sequences grouped with isolates to M. phaseolina with 100% bootstrap support. The strain XF22 has been sent to the China General Microbiological Culture Collection Center (CGMCC3.25349). Pathogenicity tests were conducted by inoculating potted plants (six plants per isolate, three times) from 45 day-old tobacco seedlings cv. Yunyan 87. Stems were randomly gently scratched with sterile needles, and a 5 mm agar disc with mycelium of the pathogen was attached to the surface of each wound, with a sterilized agar disc as control. Inoculated seedlings were incubated in growth chambers at 26℃ and 60% RH with a 12 h photoperiod/day. After ten days, symptoms that brown or black lesions on the inoculated lesions were dotted with numerous black, hard microsclerotia similar to those naturally occurring on the diseased plants, but not on the control plants. The same pathogen was re-isolated consistently, fulfilling Koch's postulates. Based on morphological, molecular, and pathogenicity test results, these isolates were identified as M. phaseolina. Charcoal rot of tobacco, caused by M. phaseolina was previously found in Guangxi in 1989 (Zhu et al. 2002), while this is the first report of M. phaseolina causing charcoal rot on flue-cured tobacco in Hunan, China. We speculate that the planting area is influenced by the preceding crop sesame. The soil carries M. phaseolina, which can cause stem rot of sesame, leading to the occurrence of tobacco charcoal rot. Our results indicated that charcoal rot caused by M. phaseolina is a new threat to flue-cured tobacco production and lue-cured tobacco might be acting as a reservoir and spreading this pathogen to other economically crops in China.

First Report of Golovinomyces bolayi Causing Powdery Mildew on Lettuce in Mexico.

In August 2022, powdery mildew symptoms were detected on lettuce (Lactuca sativa) in a commercial field located in Quecholac, Puebla, Mexico. Signs appeared as whitish powdery masses on leaves. Disease incidence was about 100% and signs covered up to 40% of leaf surface. Mycelium was amphigenous forming white patches. Hyphal appressoria were indistinct or nipple-shaped and solitary. Conidiophores (n= 30) were hyaline, erect, arising from the upper surface of hyphal mother cells or lateral, and of 90 to 201 µm long. Foot cells were cylindrical, of 49 to 92 × 10-15 µm, followed by 1-3 shorter cells, and forming conidia in chains. Conidia (n= 100) were hyaline, ellipsoid-ovoid, doliiform-subcylindrical, 27 to 40 × 14 to 20 µm. Conidial germination belonging to the Euoidium type. Chasmothecia were not observed. The morphological characters were consistent with those of Golovinomyces bolayi (Braun et al. 2019). A voucher specimen was deposited in the Herbarium of the Department of Agricultural Parasitology at the Chapingo Autonomous University under accession number UACH451. To confirm the identification of the fungus, genomic DNA was extracted from conidia and mycelium following the CTAB method (Doyle and Doyle 1990), and the internal transcribed spacer (ITS) region was amplified by PCR using the primers ITS5/ITS4 (White et al. 1990) and sequenced. The resulting 506 bp sequence had 100% identity to those of G. bolayi (LC417109 and LC417106). Phylogenetic analyses using the Maximum Likelihood and Maximum Parsimony methods were performed and confirmed the results obtained in the morphological analysis. The isolate UACH451 grouped in a clade with isolates of G. bolayi. The ITS sequence was deposited in GenBank under accession number OR467546. Pathogenicity was confirmed by gently dusting conidia onto ten leaves of healthy lettuce plants. Five non-inoculated leaves served as controls. The plants were maintained in a greenhouse at 25 to 30 ºC, and relative humidity of 70%. All inoculated leaves developed similar symptoms to the original observation after 10 days, whereas control leaves remained disease free. Microscopic examination of the fungus on inoculated leaves showed that it was morphologically identical to that originally observed. Based on morphological data and phylogenetic analysis, the fungus was identified as G. bolayi. This pathogen has been previously reported causing powdery mildew on lettuce in Argentina, Canada, Chile, Ecuador, Peru, USA and Venezuela (Braun et al. 2019; Mieslerová et al. 2020). To our knowledge, this is the first report of G. bolayi causing powdery mildew on lettuce in Mexico.

First report of the Horse nettle virus infecting tomato (Solanum lycopersicum L.) in the United States.

Tomato (Solanum lycopersicum L.), a member of the Solanaceae family, represents one of the most extensively cultivated vegetable species worldwide and traces its origin to western South America (Caruso et al. 2022). In a field survey conducted in 2023 in Bixby, Tulsa County, Oklahoma, distinct symptoms were noted in two plants: one exhibited mottling and cupping of leaves and brown discoloration on leaves, petioles, and stems, while the other displayed a downward curling of leaves. Leaf samples from both symptomatic tomato plants (labelled as K4 and K5) were collected, and total RNA was extracted individually via the TRI Reagent® method (Molecular Research Center Inc., Cincinnati, OH, USA). Subsequently, the RNA samples were pooled and subjected to high-throughput sequencing (HTS) on the NextSeq 500/550 high-output kit v2.5 (Illumina, U.S.A.) at the genomic facility, Oklahoma State University (Stillwater, OK). Total read count of 8,227,020 (average length =150.5 bp) was obtained, trimmed, and de novo assembled using CLC Genomics Workbench v22.0.1 (QIAGEN) and used for BLASTn and BLASTx analysis. Two contigs: 6,375 bp (average coverage 2,915.92, read count 142,538) and 3,564 bp (average coverage 3,035.91, read count 82,370) from the pooled sample showed 88.6% and 96.7% nucleotide identities with RNA 1 (OP292294) and RNA 2 (OP292295) of Horse nettle virus A (HNA-A) isolate MD-1, respectively. Sequences of both partial contigs (RNA 1, accession no. PP063196) and RNA 2, accession no. PP063197) were submitted to GenBank. The HTS data did not reveal any other viral or viroid sequences in these two tomato samples. To further confirm the presence of HNV-A, total RNA from K4 and K5 samples was tested individually by RT-PCR using HNV specific primers (Supplementary Table 1) based on the two partial contig sequences. The expected PCR products (491 bp and 451 bp) were obtained only from the K4 sample and none from the K5 sample. PCR products were extracted from an agarose gel, cloned into the pGEM®-T Easy vector (Promega), and transformed into Escherichia coli DH5α cells (New England Bio Labs). Two clones for each PCR product were sequenced by Sanger sequencing. Nucleotide sequence comparisons and BLASTn analysis of 491 bp and 451 bp showed 86% and 97% nucleotide identity with RNA 1 and RNA 2 of HNV-A isolate MD-1 (OP292294 and OP292295), respectively. Additionally, eight more leaf samples from eight different symptomatic tomato plants were collected in the same field and tested by RT-PCR as described above. All eight samples were positive by RT-PCR, but no PCR band was obtained in the total RNA from a healthy tomato leaf used as a control. Sequences from the PCR products were identical to the obtained HTS sequences. Our results confirmed for the first time that HNV-A can infect tomatoes. Currently, HNV-A has been reported to only infect a single weed (Horse nettle, Solanum carolinense) (Zhou et al. 2023). The identification of HNV-A in tomatoes in Oklahoma suggests a potential host shift is of concern for local growers as well as tomato growers worldwide. This shift underscores the urgency for an in-depth investigation into the transmission and host specificity of HNV-A. This is the first report in the United States and the world that HNV-A could infect tomatoes naturally in a grower field.

Colorimetric LAMP assay for detection of Xanthomonas phaseoli pv. manihotis in cassava through genomics: a new approach to an old problem.

Bacterial spot caused by Xanthomonas phaseoli pv. manihotis (Xpm) is considered the main bacterial disease that affects cassava, causing significant losses when not properly managed. In the present study, a fast, sensitive, and easy-to-apply method to detect Xpm via colorimetric loop-mediated isothermal amplification (LAMP) was developed. In order to ensure the use of a unique to the target pathovar core region for primer design, 74 complete genomic sequences of Xpm together with different bacterial species and pathovars were used for comparative genomics. A total of 42 unique genes were used to design 27 LAMP primer sets, from which nine primers were synthesized and only one (Xpm_Lp1 primer set) showed sufficient efficiency in preliminary tests. The sensitivity, assessed by a serial dilution of the type strain (IBSBF 278) DNA, yielded high sensitivity, detecting up to 100 fg. The LAMP primers showed high specificity, not cross-reacting with other bacterial species or other pathovars tested, and amplifying only the Xpm isolates. Tests confirmed the high efficiency of the protocol using infected or inoculated macerated cassava leaves, without the need for additional sample treatment. The LAMP test developed in this study was able to detect Xpm in a fast, simple, and sensitive way, and it can be used to monitor the disease under laboratory and field conditions.

First Report of Root Rot of Cathaya argyrophylla caused by Fusarium oxysporum in China.

Cathaya argyrophylla [Chun & Kuang.] is an ancient relict plant and its embryonic development is similar to that of Pinus species. This has important scientific value for studying the phylogeny of Pinaceae (Wu et al. 2023). In July 2022, root rot was detected in the seedling cultivation base of C. argyrophylla in Daozhen County, Guizhou Province, China (28.89 °N, 107.6 °E). The incidence of the disease was 30% (n = 100); the susceptible plants wilted, leaves withered, and roots showed brown-to-black lesions and rot. Ten root tissues were randomly collected from the edges of the lesions of six symptomatic susceptible plants. The tissues were sterilized with 75% alcohol for 30 seconds, followed by 2-minute immersion in 3% sodium hypochlorite. After washing with sterile water, the tissues were incubated on potato dextrose agar (PDA; BoWei, Shanghai) at 28 ℃ for five days. Four single-spore cultures were obtained using a single-spore isolation method (Gong et al., 2010). Single-spore cultures grew rapidly on PDA. After five days of incubation, the colonies were white and pink, indicating a large amount of aerial mycelia. Microconidia were ovate or ellipsoid, measuring 5.0-10.0 × 1.5-3.0 µm (n = 50); Macroconidia were falcate, slightly curved or straight, measuring 19.5-28.5 × 2.0-6.0 µm (n = 50). Based on morphological features, the pathogen was considered to be Fusarium spp. (Leslie and Summerell 2006). Three representative strains, GF5, GF6, and GF7, were selected for molecular identification, and genomic DNA was extracted to confirm morphological diagnosis. The internal transcribed spacer (ITS) (White et al. 1990) was amplified using primers ITS1/ITS4, and the ß-tubulin gene (Varga et al. 2011) was amplified using primers Bt2a/Bt2b. The ITS and ß-tubulin sequences were aligned with GenBank, and amplification of the genes from the three isolates was consistent. The ITS (OP482273) and ß-tubulin (OR825353) sequences of GF5 were stored in GenBank, and their homology with Fusarium oxysporum HC131(accession numbers MW600442 and MW670451) was 99 to 100%. Maximum likelihood analysis using MEGA 11.0 showed that isolate GF5 belongs to F. oxysporum. The reconstructed phylogenetic tree confirmed the phylogenetic position of the isolate GF5. The pathogenicity test was carried out using GF5 and GF6 isolates. The taproots of ten 3-year-old C. argyrophylla plants were washed, and then the roots were immersed in a 2 × 106/mL conidial suspension for one hour. Ten plants with sterile water were used as controls. After planting in pots (30 × 25 cm) with sterilized forest soil, the plants were cultured in a greenhouse (25 ℃ and 12-hour photoperiod). Thirty days after inoculation, all plants inoculated with the isolated pathogen showed wilting symptoms, and the roots showed typical root rot symptoms, whereas the control group showed no symptoms. The pathogens re-isolated from all inoculated plants were morphologically identical and had ITS sequences identical to F. oxysporum, validating Koch's hypothesis. The pathogenicity test was repeated twice and similar results were obtained. Although this fungus has been previously reported to cause root diseases in hosts, such as Musa nana Lour. and Pinus massoniana Lamb. (He et al. 2010; Luo et al. 2020), to our knowledge, this is the first report of F. oxysporum causing root rot in C. argyrophylla. These findings provide a basis for the development of management strategies for C. argyrophylla infection.

Survey of Heterodera glycines population abundance and virulence phenotypes during 2021-2022 in Heilongjiang Province.

Soybean cyst nematode (SCN), Heterodera glycines, poses a significant threat to global soybean production. Heilongjiang, the largest soybean-producing province in China, contributes over 40% to the country's total yield. This province has much longer history of SCN infestation. To assess the current situation in Heilongjiang, we conducted a survey to determine the SCN population density and virulence phenotypes during 2021-2022 and compared the data with a previous study in 2015. A total of 377 soil samples from 48 counties representing eleven major soybean-planting regions were collected. The prevalence of SCN increased from 55.4% in 2015 to 59% in the current survey. The population densities ranged from 80 to 26,700 eggs and juveniles per 100 cm3 of soil. Virulence phenotypes were evaluated for 60 representative SCN populations using the HG type test, revealing nine different HG types. The most common virulence phenotypes were HG types 7 and 0, accounting for 56.7% and 20% of all SCN populations, respectively. The prevalence of populations with a reproductive index (FI) greater than 10% on PI548316 increased from 64.5% in 2015 to 71.7%. However, the FI on the commonly used resistance sources PI 548402 (Peking) and PI 437654 remained low at 3.3%. These findings highlight the increasing prevalence and changing virulence phenotypes of SCN in Heilongjiang. They also emphasize the importance of rotating soybean varieties with different resistance sources and urgently identifying new sources of resistance to combat SCN.

First report of Fusarium verticillioides causing leaf rot on Artemisia argyi in China.

Artemisia argyi is a perennial herb native to East Asia. It is an important traditional Chinese medicinal plant known for its strong flavor and medicinal effects. It is rich in active ingredients and has a wide range of biological activities, including anti-inflammatory, antioxidant, and immune regulation properties. From May to July in 2023, a serious leaf rot outbreak occurred on A. argyi in several farms (approximately 200 acres) in Tanghe county (32°46'44" N, 112°43'13" E), Henan Province, China. The incidence rate reached 65% (n=200). Pale yellow spots (1-2 cm in diameter) first appeared on the leaves, then expanded to form irregular yellowish-brown lesions, eventually causing the entire leaves to wither. Diseased leaves (30) were collected and cut into 5 x 5 mm2 pieces in the areas between infected and healthy tissues. The excised plant tissues were sterilized in 75% ethanol and 1% sodium hypochlorite solution for 30 seconds and one minute, respectively. The tissues were then rinsed with sterile water and placed on potato dextrose agar (PDA) followed by incubating at 25 °C for 3 days. The isolated strains belonged to the genera Fusarium and Alternaria. After pathogenicity verification, 25 purified Fusarium strains were obtained. Three representative strains (AC-Q, AC-X, AC-Y) from different regions were used for further studies. Each strain formed abundant aerial mycelium that was initially white and later developed into purple pigments. Aerial conidiophores were sparsely branched, terminating with verticillate phialides. Macroconidia were slender, straight, and measured 21.8 to 47.5 × 3.1 to 4.4 µm, with two to four septa. Microconidia were clavate and measured 8.31 to 11.6 × 2.1 to 3.5 µm. Morphological characteristics were consistent with the species description of Fusarium verticillioides (Sacc.) Nirenberg 1976 (Leslie and Summerell, 2006). The rDNA internal transcribed spacer (ITS), ß-tubulin gene (tub2), translation elongation factor 1-alpha gene (tef1), calmodulin (cmdA), RNA polymerase II largest subunit (rpb1) and RNA polymerase II second largest subunit (rpb2) were amplified for molecular identification (O'Donnell et al., 2022). The sequences were deposited in GenBank with accession Nos. OR960548, OR960552, OR960555 (ITS), OR972413, OR972414, OR972415 (tub2), OR797685, OR797686, OR797687 (tef1), OR972410, OR972411, OR972412 (cmdA), PP035106, PP035107, PP035108 (rpb1), and PP035109, PP035110, PP035111 (rpb2). BLASTn analysis of AC-Q sequences exhibited 99 to 100% similarity with F. verticillioides sequences (strains CBS 576.78) MT010888 of cmdA, MT0109566 of rpb1, and MT010972 of rpb2. A phylogenetic tree was constructed with concatenated sequences (tub2, tef1, cmdA, rpb1, rpb2), alongside the sequences of the type strains using the neighbor-joining method. The three strains formed a clade with the type strain CBS 576.78 of F. verticillioides, and were separated from other Fusarium spp. These morphological and molecular identifications indicated that the pathogen was F. verticillioides. Pathogenicity was tested on 10 healthy 2-month-old potted seedlings by spraying them with a conidial suspension (106 conidia ml-1), and 5 seedlings were sprayed with sterilized water as a control. The plants were placed in a climate incubator at 28°C and a relative humidity of approximately 90%. Ten days after seedling inoculation, typical lesions were observed on the treated plants, except in the control group. The reisolated strains were identified as F. verticillioides by morphological and molecular characterization, fulfilling Koch's postulates. F. verticillioides is known to cause Fusarium ear rot on maize, as well as diseases on other plants in China such as Brassica rapa (Akram et al., 2020) and Schizonepeta tenuifolia (Li et al., 2024). This is the first report of F. verticillioides causing leaf rot on A. argyi worldwide. Identification of the pathogen is crucial for implementing management approaches to reduce yield losses.

Effects of hybrid, foliar treatments, and infection conditions on bacterial leaf streak of sweet corn.

Bacterial leaf streak (BLS) of corn caused by Xanthomonas vasicola pv. vasculorum was first reported in the United States in 2017. The biology and management of BLS are poorly understood. The objective of this work was to determine the effects of hybrid, foliar treatments, and infection conditions (timing, temperature, inoculation site) on BLS of sweet corn. Field studies were conducted to determine if hybrid or foliar disease management treatments influenced BLS development and yield. Corn leaves were inoculated in plots with X. vasicola pv. vasculorum, and noninoculated plots were used for comparison. The leaf incidence and severity of BLS differed significantly among sweet corn hybrids, suggesting different levels of susceptibility to BLS. Grain yield was significantly reduced (14.7%) by BLS for one hybrid. The corn growth stage at time of infection influenced BLS, with incidence and severity significantly greater following inoculation at stage V6 than V9. Foliar application of Kocide®, LifeGard®, and Kocide®+LifeGard® significantly reduced leaf severity compared to nontreated controls in field studies. Kocide® significantly reduced leaf incidence, but no treatments significantly increased yield vs. controls. In comparisons of inoculation methods in a growth chamber, lesion length on leaves was significantly greater on stalk-inoculated than leaf-inoculated plants. Lesions developed on leaf-inoculated plants only at inoculation sites whereas lesions developed on stalk-inoculated plants on multiple leaves. In controlled environments, lesion length on leaves was significantly greater at 21°C than 27°C and 32°C. This study expands our understanding of factors that influence development and management of BLS of sweet corn.

First Report of White Mold Caused by Sclerotinia sclerotiorum on Hymenocallis glauca in Mexico.

In Mexico, there are 29 native species of the genus Hymenocallis, where H. glauca is one of the most cultivated bulbous plants. It holds economic importance as it is commercialized as a potted plant and cut flower (Leszczyñska and Borys, 2001). In October 2023, field sampling was conducted in the Research Center in Horticulture and Native Plants (18°55'55" N, 98°24'02.8"W) of UPAEP University. H. glauca diseased plants were found in an area of 0.4 ha, with an incidence of 35% and an estimated severity of 45% on infected plants in vegetative stage. The symptoms included chlorosis of foliage, necrosis at the base of the stem, and soft rot with abundant white to gray mycelium and abundant production of black, irregular sclerotia of approximately 3.5 mm diameter. Finally, the plants wilted and died. The fungus was isolated from 40 symptomatic plants. Sclerotia were collected, disinfested with 3% NaOCl for one minute, rinsed with sterile distilled water (SDW), and plated on Petri dishes containing potato dextrose agar (PDA) with sterile forceps. Subsequently, a sterile dissecting needle was used to place fragments of mycelium directly on Petri dishes with PDA. Plates were incubated at 23 °C in dark for 7 days. One isolate was obtained from each diseased plant by the hyphal-tip method (20 isolates from sclerotia and 20 from mycelium). After 7 days, colonies had fast-growing, dense, and cottony-white aerial mycelium forming irregular sclerotia of 3.57 ± 0.59 mm (mean ± standard deviation, n=100). In each Petri dish there were produced 21.5 ± 7.9 sclerotia (mean ± standard deviation, n=40), after 11 days; these were initially white and gradually turned black. The isolates were tentatively identified as Sclerotinia sclerotiorum based on morphological characteristics (Saharan and Mehta 2008). Two representative isolates were chosen for molecular identification and genomic DNA was extracted by the CTAB protocol. The ITS region and the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene were amplified and sequenced (Staats et al. 2005; White et al. 1990). The sequences of a representative isolate (SsHg3) were deposited in GenBank (ITS- PP094578; G3PDH- PP101843). BLAST analysis of the partial sequences ITS (519 bp), and G3PDH (950 bp) showed 100% similarity to S. sclerotiorum isolates (GenBank: MG249967, MW082601). Pathogenicity was confirmed by inoculating 30 H. glauca plants in vegetative stage grown in pots with sterile soil. Ten sclerotia were deposited at the base of the stem, 10 mm below the soil surface. As control treatment, SDW was applied to 10 plants. The plants were placed in a greenhouse at 23 °C and 90% relative humidity. After 17 days, all inoculated plants displayed symptoms similar to those observed in the field, while no symptoms were observed on the controls. The fungus was re-isolated from the inoculated plants as described above, fulfilling Koch's postulates. The pathogenicity tests were repeated three times. S. sclerotiorum has been reported causing white mold on other bulbous plants, like fennel (Foeniculum vulgare) in Korea (Choi et al. 2015). To our knowledge, this is the first report of S. sclerotiorum causing white mold on H. glauca in Mexico. Information about diseases affecting this plant is very limited, so this research is essential for developing integrated management strategies and preventing spread to other production areas.