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.

First report of leaf spot caused by Paramyrothecium roridum on Coffea arabica in Hawai'i, USA.

During the 2022-2023 season, the harvested coffee crop in Hawai'i (Coffea arabica) was valued at $57.1 million (USDA NASS 2023). In September 2022, coffee leaf samples with foliar leaf spots affecting the Kona Typica variety were collected from Honaunau, Hawai'i, incidence <10%. The symptoms were circular, necrotic leaf spots with yellow margins, which merged, resulting in complete leaf blade coverage and subsequent leaf drop. Sporodochia were present on the abaxial leaf surface. Symptomatic leaf tissue was disinfected in 10% bleach solution for 60 seconds and chlorotic leaf tissue from the spot margins were excised and placed onto water agar and potato dextrose agar (PDA; Difco, USA). After a 7-day incubation period, pure cultures with white aerial mycelium having sporodochia arranged in concentric rings with olivaceous to black conidial masses were isolated. The conidia were aseptate, hyaline, smooth, cylindrical with rounded ends, measuring 5.1 to 6.8 µm long and 1.7 to 2.3 µm wide (n=50). Based on symptomology and cultural/morphological characteristics (Huaman-Pilco et al. 2023; Lombard et al. 2016; Pelayo-Sanchez et al. 2017), the isolates were initially identified as Paramyrothecium roridum (Tode) L. Lombard & Crous, comb. nov. (syn. Myrothecium roridum Tode). Fungal identification of isolate P22-81-2 was further confirmed using BLAST analysis of bulk sequenced PCR products of the ribosomal DNA internal transcribed spacer (ITS) region (White et al. 1990), ß-tubulin (ßtub), RNA polymerase II (RPB2), and calmodulin genes (Lombard et al., 2016; Huaman-Pilco et al., 2023). The gene sequences (GenBank accession nos. PP211198, PQ192517-19) were >98.4% identical to the P. roridum type specimen (CBS 357.89). A multilocus maximum likelihood phylogenetic analysis incorporating sequence data from previous relevant studies (Lombard et al., 2016; Pinruan et al. 2022) confirmed species identification. To prove pathogenicity, four, 26-month-old Kona Typica variety seedlings were foliar inoculated with a 1 X 106 conidia/ml suspension using a perfume atomizer. An additional four plants were inoculated in a similar manner with sterile water which served as controls. All plants were sprayed to drip on both the upper and lower leaf surfaces and incubated in a clear plastic bag to keep the humidity levels between 90 to 100% for 48 hours at 24°C. After 48 hours, the plants were removed from the bags, placed on a greenhouse bench, and observed weekly for symptom development. Within seven days light brown sunken spots had developed on all inoculated plants. The spots continued to enlarge having a dark distinct margin, light tan center, chlorotic halo, and formed concentric rings, which were identical to the original diseased samples. Leaf spots were not present on any of the control plants. The test was conducted twice. A fungus was consistently reisolated from the leaf spot margins of inoculated plants and morphologically (PDA) and molecularly (ITS, ßtub, RPB2, calmodulin) identified as P. roridum, thus fulfilling Koch's Postulates. To the best of our knowledge, this is the first report of P. roridum causing leafspots on C. arabica plants in Hawai'i. This pathogen has been reported on coffee in other parts of the world including Colombia, Costa Rica, Guatemala, Puerto Rico, and Mexico (USDA Fungus-Host Database). Under the right conditions, P. roridum has the potential to cause leafspots and defoliation resulting in economic losses for coffee growers in Hawai'i.

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.

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.

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.

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.

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.

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.

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 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.

Development of a recombinase polymerase amplification method combined with a lateral flow dipstick assay for rapid detection of the larch pathogen Neofusicoccum laricinum.

Neofusicoccum laricinum, an important pathogenic species, causes shoot blight of larch. In China, large areas of Larix principis-rupprechtii forests are threatened by this pathogen. Currently, this pathogen is on the list of quarantine pests in Chinese. Due to the widespread and severe damage caused by N. laricinum, a reliable and accurate diagnostic tool is urgently needed. In this study, we first identified a Nlar12009 as a N. laricinum-specific gene through genomic sequence data and bioinformatic analysis. Specific primer pairs and DNA probes were designed to detect the target pathogen using a novel recombinase polymerase amplification assay with a lateral flow dipstick (RPA-LFD) method. We optimized the RPA-LFD assay to ensure high specificity to N. laricinum. Our results showed that the assay exclusively detected N. laricinium isolates with no cross-reaction with other isolates of fungaland oomycete species and nematodes. Furthermore, our detection technique exhibited a 10-fold higher sensitivity (10 fg/mL) than conventional polymerase chain reaction (PCR) for N. laricinum detection. Our developed RPA-LFD assay is proved to be a highly specific, sensitive, time-saving, and convenient method for the diagnosis of N. laricinum and shows great potential in field application.

Pyrrolnitrin in Pseudomonas chlororaphis strain AFS009 metabolites reduces constitutive and DMI-induced MfCYP51 gene expression in Monilinia fructicola.

Brown rot, caused by Monilinia fructicola, is one of the most economically important diseases of peach. Demethylation inhibitor (DMI) fungicides play an important part in managing brown rot in the southeastern U.S., but over the last 20 years, reduced efficacy to DMIs has been reported in field isolates overexpressing the DMI target enzyme encoding MfCYP51 gene. Metabolites of the biocontrol agent (BCA) Pseudomonas chlororaphis strain AFS009 suppressed the MfCYP51 gene in sensitive and resistant M. fructicola isolates previousely, but it is not known what molecule was responsible. The goals of this study were to determine the presence and role of pyrrolnitrin (PRN), a common metabolite of P. chlororaphis and chemical analogue to fludioxonil with antifungal activity, in the suppression of the MfCYP51 gene and to investigate if MfCYP51 expression can also be suppressed by Bacillus subtilis. High-performance liquid chromatography (HPLC) detected pyrrolnitrin at 1.75 µg/mg in P. chlororaphis metabolites formulated as Howler EVO (Howler). PRN at 0.1 µg/ml, fludioxonil at 0.1 µg/ml, and Howler applied at a dose that contained 0.1 µg/ml PRN significantly reduced the MfCYP51 gene expression at similar levels in DMI-resistant isolates. Furthermore, MfCYP51 expression in DMI-sensitive and three DMI-resistant isolates treated with Howler (88.1 µg/ml), Theia (209.5 µg/ml), propiconazole (0.3 µg/ml), or the mixture of either Howler or Theia + propiconazole revealed that Howler significantly reduced the MfCYP51 target gene expression in two of three sensitive and all three resistant M. fructicola isolates. On the other hand, Theia showed no suppressive effect and even increased the MfCYP51 gene expression level in two of three resistant isolates. In detached fruit assays on apple with a DMI resistant isolate, only the mixture of Howler + 50 µg/ml propiconazole resulted in synergism. The results indicate that suppression of MfCYP51 target gene is BCA-dependent and can be induced by pyrrolnitrin.

First report of Fusarium falciforme causing root and stem rot in cowpea (syn. black-eyed pea; Vigna unguiculata) in California.

In both April 2018 and September 2019, cowpeas / black-eyed peas (Vigna unguiculata) in one field in Tulare County, California were observed with tap root rot, both underground (foot) and aboveground stem rot, and in some cases canopy decline, compromising bean formation. In both fields, < 5% of plants appeared affected. Foot and stem segments (~1 cm) of 5-10 plants / field were disinfested sequentially with 0.1% Tween 20 (dip), 70% ethanol for 30 s, and 1% sodium hypochlorite for 2 min and placed on 1:10 potato dextrose agar with 0.03% tetracycline and Fusarium selective medium (Leslie and Summerell 2006). Fusarium-like isolates (dominant in isolation plates) were transferred to 0.6% KCl agar, where fusiform, curved macroconidia and varied microconidia in false heads on elongated monophialides were observed, characteristic of the Fusarium solani species complex (FSSC) (Leslie and Summerell 2006). Isolates CS221, CS222, and CS520 (representing different plants and locations) were saved as single hyphal tip cultures. An Illumina-derived genome sequence was assembled (Burkhardt et al. 2019) and partial tef1ɑ and rpb2 sequences (O'Donnell et al. 2022) were extracted from genome sequences in silico. Sequences were 99.9-100% identical to one another and to deposited F. falciforme isolates based on Fusarium ID and Fusarium MLST for tef1ɑ and rpb2, respectively (tef1a accessions: NRRL 28562 and NRRL 32331; rpb2 accession: NRRL 22857), and were deposited in GenBank (accessions in supplementary table). Pathogenicity was evaluated in three-week-old cowpea plants (cv. CB46rk2) in the greenhouse (13.5-33.6℃; 12:12 h L:D). The tap root / stem was wounded (1 mm wide, 2 mm deep) ~ 2 cm below the soil line and drenched with 50 ml of 106 spores / ml 0.1% water agar or with 0.1% water agar (negative control). The trial was arranged in a Randomized Complete Block Design with three blocks and 2-3 plants / isolate / block, and conducted twice. 52 d post-inoculation, below ground tap root / stem rot developed in 83% of F. falciforme-inoculated plants, with lesion lengths ranging from 25.2 ± 4.2 to 29.2 ± 8.0 mm (P = 0.893 for isolate, ANOVA). Canopy decline developed in 33-50% of plants across treatments in trial 1 (P = 0.859 for isolate) but not in trial 2, likely due to cooler conditions in trial 2 (January-March) vs. trial 1 (May-July), which were less stressful. F. falciforme isolates did not affect bean biomass (dry weight) vs. negative controls (12.5-14.8g / plant; P = 0.949 for pathogen treatment). FSSC isolates were recovered from 100% of symptomatic plants in the inoculated treatments but not in negative controls (both trials) and representative isolates from all treatments were confirmed as F. falciforme (tef1a analysis; trial 2 only). This study establishes F. falciforme as a root and stem rot pathogen of cowpea in California-a disease previously attributed to the morphologically and phylogenetically distinct F. phaseoli (syn. F. solani f. sp. phaseoli), but which lacked modern etiological studies (Frate et al. 2018; Geiser et al. 2021). This work is consistent with previous reports of F. falciforme as a root / stem rot pathogen in cowpea (Ajamu et al. 2023) and other beans (Sousa et al. 2017; Duarte et al. 2019). Clarification of disease etiology will improve accurate diagnosis and effective crop rotation-based management, since F. falciforme is also a pathogen of other California crops including melon, tomato and pistachio.

First Report of Leaf Rot on Catalpa bungei caused by Fusarium verticillioides in China.

Catalpa bungei originates from China. Because of its well-developed root system and strong resistance to wind and soil, it is one of the top ten recommended species of ecological management (Jian et al., 2022). In September 2023, a severe leaf rot of C. bungei was observed at Lanlake farm (500 acres) in Nanyang (33°3'23" N, 112°28'50" E), China. The incidence rate of leaf rot reached 45% (n = 100). The pale-yellow spots initially appeared on the adaxial surface of leaf margins, subsequently enlarging to form irregular black rot lesions, with the yellow halos around the necrotic area of the lesion, ultimately causing the entire leaves to wither. Diseased leaves (20) were collected, cut into pieces, sterilized, and then placed on potato dextrose agar (PDA). A total of 25 purified fungal strains were isolated, and three strains (QS2-1, QS2-2, QS2-3) from distinct areas were selected for further analysis. Each strain produced abundant aerial mycelium, initially white, which later developed purple pigments. The aerial conidiophores were sparsely branched, ending with verticillate phialides. The strains generally produced many more microconidia than macroconidia on PDA media. Microconidia were clavate and measured 3.9 to 6.6 × 1.1 to 2.4 µm (n = 50). To produce macroconidia, we used YPG liquid medium (0.3% yeast extract, 1% peptone, and 2% glucose) with shaking (200 r.p.m.) for 5 days. Macroconidia were slender, straight, and measured 19.5 to 27.1 × 1.9 to 3.5 µm, with 3 to 5 septa (n = 50). The morphological characteristics matched 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 submitted to GenBank with accession numbers OR741762, OR741763, OR741765 (ITS), OR762222, OR762223, OR939807 (tub2), OR939799, OR939800, PQ035927 (tef1), OR778611, OR939808, OR939809 (cmdA), PQ035921, PQ035922, PQ035923 (rpb1), and PQ035924, PQ035925, PQ035926 (rpb2). BLASTn analysis of QS2-1 sequences exhibited 99% similarity with F. verticillioides sequences (strains CBS 576.78) MT010888 of cmdA (711/713, 99%), MT010956 of rpb1 (1790/1791, 99%), and MT010972 of rpb2 (868/870, 99%). A phylogenetic tree was constructed using concatenated sequences along with the sequences of the type strains employing the neighbor-joining method, showing the three strains formed a clade with the type strain CBS 576.78. Pathogenicity was tested on 10 healthy potted seedlings by spraying them with a conidial suspension (106 conidia ml-1), while 5 seedlings were sprayed with sterilized water as a control. The plants were placed in climate incubators. Ten days after inoculation, typical lesions were observed on the treated plants, but not on the control group. The reisolated strains were identified as F. verticillioides through morphological characterization, thus fulfilling Koch's postulates. F. verticillioides is known to cause Fusarium ear rot on maize, and other plants including Brassica rapa (Akram et al., 2020) and Schizonepeta tenuifolia (Li et al., 2024). This is the first documented instance of F. verticillioides causing leaf rot on C. bungei globally. Identifying the pathogen is critical to implementing effective disease management strategies, especially in choosing proper pesticide agents and screening disease-resistant varieties.

Characterization, Phylogenetic Analyses, and Pathogenicity of Eutiarosporella dactylidis on Sorghum in China.

Sorghum, the fifth-largest cereal crop globally and a C4 crop, mainly grows in arid and semi-arid areas. In 2021-2023, a new foliar disease of sorghum occurred in China. The diseased leaves showed water-soaked symptoms in the leaf tip and margins, resulting in half- and full-leaf desiccation and necrosis, thus affecting plant photosynthesis. A total of 24 Eutiarosporella strains were isolated from symptomatic leaves. Based on morphological characteristics and multi-locus phylogenetic analysis involving ITS, LSU, and EF1-α sequences, and the pathogenicity test, the pathogen of sorghum causing leaf blight in China was identified as Eutiarosporella dactylidis. The virulence of all E. dactylidis strains was evaluated using the spray-mycelium method. Different strains showed significantly different pathogenicities toward a susceptible cultivar, Longza 10, with disease indexes ranging from 23.76 to 60.37. This study first reported leaf blight of sorghum caused by E. dactylidis and named it "Eutiarosporella leaf blight", which provides a theoretical basis for farmers in disease management.

First Report of Cacao Mild Mosaic Virus Associated with Cacao (Theobroma cacao) in Hawai'i, USA.

Cacao, Theobroma cacao, is an important tropical agricultural crop and the key ingredient of chocolate, which has an international trade value of $103 billion/year (Marelli et al. 2019). Cacao mild mosaic virus (CaMMV) is a badnavirus that causes mild symptoms compared with some of the closely related species of Cacao swollen shoot virus (CSSV), the latter of which are currently only found in West Africa (Marelli et al. 2019; Ullah et al. 2021). CaMMV was recently found in symptomatic commercial cacao trees in Mayagüez, Puerto Rico (Puig et al. 2020), and subsequently detected in a USDA ARS (Agricultural Research Service) quarantine greenhouse in Miami, FL (Puig 2021). The USDA ARS germplasm repository in Hilo, Hawai'i, USA serves as a backup collection for Puerto Rico's cacao germplasm, and field trials have been established from cacao germplasm from Miami to evaluate select varieties. To determine if CaMMV is present in the collection in Hilo, greenhouse and field accessions were tested. Using an optimized sampling and PCR protocol established by Puig (2021), three young cacao petioles per tree or seedling were collected and pooled, and DNA was extracted using the NucleoSpin Plant II commercial kit (Macherey-Nagel, Düren, Germany) following the manufacturer's instructions. Samples were molecularly identified via end point PCR, gel electrophoresis, and Sanger sequencing. PCR amplification of CaMMV using the virus-specific primer set Mia-1396F (5'-ACCGTGTCTAYCAGCACTGGA-3') and Mia-1667R (5'-GACCACCGTCAGCCAGAC-3') produced 289 bp amplicons. Of 230 plants sampled, 26 CaMMV positive detections were discovered in greenhouse and field plantings in Hilo. Most CaMMV+ plants contained some form of leaf chlorosis (96.2%). The sequenced PCR products from Hawai'i were deposited in GenBank (accession nos. OQ692890-OQ692891) and showed 99.2% nucleotide identity to CaMMV accessions from Puerto Rico (MW052520; n = 23) and 98.1% nucleotide identity to CaMMV accessions from Florida, USA (MZ409692; n = 3) in BLASTn analysis. For species-level confirmation, the RT-RNase H domain was amplified from 9 isolates using CaMMV-specific primers (Mia5385F, 5'-AGGACAACGGCTTTCTTGGT-3'/Mia6616R, 5'-GAGACTAACTTGGTTAGGGCT-3'), sequenced, and deposited in GenBank (accession nos. PP997461-PP997462). Sequences matched most closely to GenBank isolates from Puerto Rico (MT253659; 98.0%; n = 7) and Trinidad and Tobago (NC_033738; 97.1%; n = 2). CaMMV, previously known as cacao Trinidad virus A, was first reported in 1943 and was associated with 7 to 33% yield reduction, loss of vigor, and tree decline (Posnette 1944; Swarbrick 1961). CaMMV is known to exist in the Americas and was most recently detected in Brazil and Indonesia (Kandito et al. 2022; Ramos­Sobrinho et al. 2021). To the best of our knowledge, this is the first report of CaMMV infecting cacao in Hawai'i. The cacao industry continues to expand in Hawai'i, and cultivation occurs on at least four of the main islands including Hawai'i Island, O'ahu, Maui, and Kaua'i. To develop disease management strategies, further investigation is needed to define CaMMV symptomology, and determine the distribution and effect this virus has on production in Hawai'i. In the meantime, cacao will be screened regardless of visible symptomology to maintain pathogen-free accessions and avoid the transfer of virus-containing germplasm.

Chemical management strategies for halo blight of hop and in vitro sensitivity of Diaporthe humulicola populations to various fungicide classes.

Halo blight of hop, caused by Diaporthe humulicola, has increased in eastern North America since 2018. When left untreated, the disease can cause yield loss ranging from 17-56%. Currently, there are no fungicides registered for use on halo blight of hop. From 2020 to 2022 field trials were conducted using 10 fungicides registered for use on powdery and downy mildew of hop to determine their efficacy against halo blight. To validate field results, the EC50 value was determined for each active ingredient including flutriafol, tebuconazole + fluopyram, cyflufenamid, and trifloxystrobin + salicylhydroxamic acid (SHAM). Each fungicide tested had an EC50 value less than 50 ppm. A discriminatory dose was used to test the sensitivity of 206 D. humulicola isolates collected from the eastern U.S. and Canada in a poison agar assay. Results showed that tebuconazole + fluopyram decreased the incidence and severity of halo blight in the field. Also, this fungicide combination had EC50 values of 2.26 x 10-1 ppm and significantly reduced the growth of most of the isolates tested. Trifloxystrobin + SHAM decreased the presence of halo blight in the field tiral, but some isolates were less sensitive in discriminatory dose testing. Our results show that fungicides in FRAC groups 3, 7, and 11 were the most effective to control halo blight. Analyses of field trials showed a positive correlation between the severity of early season downy mildew infections and late season halo blight infections.

Nectriaceae species associated to root rot of nursery and young Eucalyptus smithii trees in Uruguay with Ilyonectria charruensis as novel species.

Forestry constitutes an important agronomical activity in Uruguay, involving the cultivation of exotic trees mainly for cellulose pulp production with Eucalyptus species. Over the last decade, E. smithii emerged as a species of interest for cellulose pulping. However, its rapid expansion has coincided with high mortality rates among young trees ranging from 5 to 85%, especially during the first and second summer after plantation. Disease surveys conducted on nine E. smithii commercial fields and three nurseries in southern and eastern Uruguay, yielded a collection of 25 isolates from E. smithii root rot belonging to the Nectriaceae family. In this study, we aimed to identify and characterize these isolates employing phenotypical and molecular studies and to assess their pathogenicity on E. smithii seedlings. Based on morphological features, the Nectriaceae isolates were subdivided into two groups, one resembling Calonectria (n=15) and another Cylindrocarpon-like (n=10). DNA sequences of the partial histone H3 (his3), actine, calmodulin, RNA polymerase II second largest subunit, translation elongation factor 1-alpha (tef1) and ß-tubulin (tub2) genes were amplified for Calonectria, as well as partial his3, tef1, tub2 and internal-transcribed spacer and intervening 5.8S (ITS) for the Cylindrocarpon-like group. Based on phylogenetic analysis and phenotypical features three species were identified and characterized; Calonectria pauciramosa (n=15), Dactylonectria novozelandica (n=2), and a novel taxon which we describe here as Ilyonectria charruensis sp. nov. (n=8). The pathogenicity trials revealed that isolates from the three species significantly reduced both shoot and root dry weights of inoculated E. smithii seedlings compared to control plants.

First Report of Botryosphaeria dothidea Causing Leaf Blight on Parrotia subaequalis in China.

Parrotia subaequalis is of great ornamental value due to its unique bark, featuring interesting textures and colors, and its large, striking galls. These characteristics make it a popular choice for bonsai cultivation. (Yan et al. 2022) . In July 2023, an outbreak of leaf blight was observed on 40, six-month-old P. subaequalis seedlings in Anqing, Anhui, China, with an incidence rate of 80%. In the early stages of infection, small brown spots appear on the leaf surface, which gradually become round or irregular and darken to a deep brown color. As the disease progresses, the affected areas expand from the leaf margins towards the center, causing the leaf surface to become concave, wilt, and necrotize. This resulted in restricted plant growth, and in severe cases, partial or complete plant death. For isolation, 30 tissue slices (5 × 5 mm) were taken from the leaves of 10 symptomatic seedlings and surface sterilized with 75% ethanol for 5 seconds, followed by five rinses with sterilized distilled water. After two days of dark incubation at 28°C, hyphal tips of fungi were transferred onto new potato dextrose agar (PDA) plates and incubated until conidia production. Six unidentified isolates with similar morphological characteristics were obtained. The colonies, initially white, darken to black after 7 to 10 days of incubation. They produced colorless, aseptate conidia that were oblong or fusiform, measuring 18-26 µm in length and 5-8 µm in width (n=50). The morphological characteristics of the isolates resembled those of Botryosphaeria (Udayanga et al. 2015) . Isolate IS2116-1 was further confirmed through molecular methods. The rDNA internal transcribed spacer (ITS) region, translation elongation factor 1-α (TEF1-α), and beta-tubulin (TUB2) genes were amplified and sequenced using the primers ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R, and Bt2a/Bt2b (Ferreira et al., 2021; Carbone et al., 1999), respectively. BLAST analysis revealed that the ITS (OR958722) sequence was 100% similar to the B. dothidea isolate HZ5(MH329650.1), TEF1-a (PP214058) sequence was 100% similar to the B. dothidea strain JZB310220(ON890458.1), and strain TUB2 (PP214057) sequence was 99.78% similar to the B. dothidea strain L14 (KR260833.1). Maximum likelihood analyses were performed for the combined ITS、TUB2、TEF datasets using PhyloSuite v1.2.2, the resulting phylogenetic tree indicated that isolate IS2116-1 clustered together with Botryosphaeria dothidea in a clade with 97% bootstrap support(Zheng et al. 2020) . Pathogenicity tests were conducted on 3-6 month-old P. subaequalis seedlings (n = 5) grown in a greenhouse. A conidial suspension (106 spores/ml) collected from the isolates was sprayed onto P. subaequalis seedlings, while the control was treated with distilled water. All plants were maintained in a growth chamber at 28°C with a 12-h photoperiod. The experiment was conducted twice independently . After 20 days of inoculation, brownish lesions similar to those observed in the field appeared on the treated plants, while the noninoculated control plants remained symptomless. The pathogen was reisolated from the leaves of the obviously diseased seedlings and confirmed as B. dothidea through morphological and sequence analysis. No isolates were obtained from uninoculated control plants, thus fulfilling Koch's hypothesis. This report marks the first record of B. dothidea causing leaf blight in P. subaequalis. In light of the rarity of natural P. subaequalis populations, it is imperative to assess both the extent of disease spread and its economic impact. These insights are crucial for devising strategies to protect this endangered species from disease threats and to preserve its ecological significance.

A qPCR assay for specific detection of Pseudomonas cannabina pv. alisalensis.

Pseudomonas cannabina pv. alisalensis is a gram-negative bacterium that causes bacterial leaf blight in Brassica crops, an important disease that could bring severe damage to the host plants. The aim of this study was to develop a tool that can reliably and accurately quantify P. cannabina pv. alisalensis and distinguish it from other closely related bacterial pathogens. Two species and six pathovars of Pseudomonas were tested: three pathovars, P. syringae pv. coriandricola, P. syringae pv. philadelphi, and P. syringae strains from Vicia faba were found or confirmed to be members of P. cannabina based on the multi-locus sequence analysis and rep-PCR results. The qPCR assay was evaluated for specificity and examined for detection limit in pure bacterial cells and bacteria-spiked plant samples. The assay was applied in monitoring the quantities of the P. cannabina pv. alisalensis DNA over time in inoculated turnip green leaves. As results, the newly developed qPCR assay detected the target DNA in P. cannabina pv. alisalensis suspension as low as 100 CFU/ml and did not detect any of the nontarget bacteria. The qPCR assay detected P. cannabina pv. alisalensis in all the inoculated samples at least 5 days before the symptoms became visible; bacterial quantity increased significantly in the first three days after inoculation but slowed down afterwards. The new qPCR assay for P. cannabina pv. alisalensis detection will facilitate early detection and disease diagnosis, assist research to provide epidemiological insights for the pathogen, and guide implementation of strategies to manage disease and prevent its spread.