Chlorotic Lesions Research Articles

First Report of Bidens Mottle Virus Causing Mild mottle and Leaf Distortion in Stachytarpheta jamaicensis in Taiwan.

Stachytarpheta jamaicensis, a traditional herbal pharmacological plant in the Family Verbenaceae that produces purplish-blue flowers, is mainly used as a garden plant in tropical and subtropical areas, including Taiwan. A begomovirus, stachytarpheta leaf curl virus (StaLCV) that caused disease on S. jamaicensis, has been reported (Xiong et al. 2005). In 2021, five symptomatic plants with mild mottle and leaf distortion (Fig. S1A, B) and three asymptomatic plants were collected in Taichung City, Taiwan. Polymerase chain reaction (PCR) and reverse transcription (RT)-PCR assays using degenerate primer pairs with expected amplicon sizes of 1.2-1.3 kb (PAL1v1978/PAR1c715; Rojas et al. 1993), 312 bp (dTospo-F2/dTospo-R2; Huang et al. 2018), and 600-750 kb (Hrp5/Pot1; Chen et al. 2006, Colinet and Kummert 1993) for Begomovirus, Orthotospovirus, and Potyvirus, respectively were performed using total DNA and total RNA plant extracts. Results showed the expected fragments were only amplified from the 5 symptomatic plants using Potyvirus degenerate primers. Three out of five randomly picked amplicons, coding the 3'-end of nuclear inclusion b protein (NIb) and 5'-end coat protein (CP) genes, were cloned and sequenced with the ABI3730 automatic sequencer (Applied Biosystems, Hammonton, NJ, USA) in Biotechnology Centre DNA-sequencing facility at National Chung Hsing University (NCHU). After NCBI BLASTN analysis, the sequences were shown to be most closely related to bidens mottle virus (BiMoV). The nucleotide sequence identities analyzed using the CLUSTAL W Methods of MegAlign program (DNASTAR, Inc., Madison, WI, USA), showed the three amplicons shared 95.8-99.8% to each other and 94.3-97.1% with 18 BiMoV isolates available in NCBI GenBank. Further RT-PCR with a specific primer (FJJ2021-278) designed from the CP of previously amplified amplicons, paired with oligo d(T) primer, were used for amplification of the 3'-CP gene and 3'-untranslated region (UTR) from total RNAs purified from symptomatic plants. The full-length CPs (804-nt and 268-aa) of the BiMoV isolates described here (Acc. Nos. OM406329 and OM406330; designated as isolate Stachy3 and Stachy7, respectively) shared 96.5-98.5% nucleotide and 97.8-99.3% amino acid identity to other BiMoV isolates. The isolate used for back-inoculation to S. jamaicensis was selected after the completion of triple single chlorotic local lesion isolation in Chenopodium quinoa. Two mechanically-inoculated S. jamaicensis plants exhibited symptoms 14-16 days post-inoculation similar to those observed in field plants and tested positive in RT-PCR using BiMoV-specific primers. In transmission electron microscopy, crude sap extracted from mechanically-inoculated C. quinoa and stained with uranyl acetate (UA) revealed flexuous filamentous virions of approximately 720 × 12 nm (Fig. S1C). A western blot assay using BiMoV antiserum (Chen and Lee 2012) revealed bands of about 34 KDa only from the mechanically-inoculated C. quinoa and the five symptomatic S. jamaicensis plants collected from the field. Taken together, we believe this is the first report of BiMoV infecting and causing mild chlorotic mottle and leaf distortion on S. jamaicensis. S. jamaicensis may serve as a new alternative host of BiMoV that can spread the disease, and consequently may directly impact the producers of horticultural or economical crops, such as lettuce, calendula, sunflower, lisianthus, and garland chrysanthemum in Taiwan.

First Report of Mulberry Rust Caused by Cerotelium fici on Morus nigra in Brazil.

Leaf rust caused by Cerotelium fici (Cast.) Arth. is the main disease affecting Moraceae family plants, such as Ficus and Morus species (Galleti and Rezende 2016; Srikantaswamy et al. 2006). In August 2020, rust symptoms were observed in 100% of mulberry (Morus nigra L.) trees in an experimental orchard (Piracicaba, SP, Brazil; 22°42'28"S, 47°37'42"W). Mulberry leaves with high rust severity became yellowish and fell-off prematurely. Pustules were light brown with yellowish halo and presented mean size of 0.9 mm2. Uredinial paraphyses (n = 50) measured 42.2 ± 0.67 µm long with wall uniformly ca 0.6-1.1 μm thick. Urediniospores were brownish, echinulate, globoid to broadly ellipsoid, and measured 27.1 ± 0.29 × 21.0 ± 0.27 µm with a wall thickness of 0.6 ± 0.01 µm (n = 100). The morphology of the urediniospores observed in this study was similar to that reported in the literature for C. fici on Morus alba and Ficus spp. (Gupta et al. 1994; McKenzie 1986; Hennen et al. 2005). We used a low-coverage genome-skimming approach to retrieve genetic information of the rRNA cluster and the mtDNA. Genomic DNA was extracted from 3-4 mg of stored urediniospores at -80 °C, macerated in liquid nitrogen, using a modified cetyl trimethylammonium bromide extraction procedure (Lo Piccolo et al. 2012), and sequenced with 150-bp paired-end reads on Illumina NovaSeq 6000 System. Raw data, (45,761,957 X 2 reads) were assembled with SPAdes v3.15.1 (Bankevich et al., 2012) and the output used to create a custom BLAST database. Loci used for the phylogenetic analyses were identified by BLASTn using, as a query, sequences of C. fici from Ficus sp. from Australia publicly available: Accession No. MH047210.1 for the rRNA and MW036502.1 for COX3. The retrieved sequences were deposited in GenBank under accession numbers OM296992 and OP797407 for the partial rRNA cluster and COX3, respectively. The Bayesian inference phylogenetic analysis of the three concatenate loci (18S, 28S, and COX3) revealed that the isolate obtained in this study (MN1) was clustered in a well-supported clade with C. fici type species. Pathogenicity tests were conducted using mulberry potted plants under greenhouse conditions (25 ± 5 °C). The urediniospores suspension (5 × 104 urediniospores ml-1) with 0.05% Tween 20 was sprayed with an airbrush on fully expanded leaves until run-off. As a control, mulberry plants were sprayed with distilled water and kept under the same conditions. Inoculated and mock-inoculated plants were kept in a dark moist chamber at 23 °C (± 2 °C) for 24 h. After this period, plants were moved to the greenhouse. The experimental design was completely randomized with five replicates, each replicate consisted of one potted plant and the experiment was performed twice. At 12 days post-inoculation, all inoculated plants showed rust symptoms identical to those observed in the field, whereas control plants had no symptoms. The first symptoms were small pustules on the abaxial surface of fully expanded leaves. Small chlorotic lesions were observed on the adaxial leaf surface, which evolved into necrotic lesions. The pathogen was re-inoculated into potted plants, where it was maintained through monthly inoculations. To our knowledge, this is the first report of mulberry rust on M. nigra in Brazil. As mulberry leaves are the only natural food for silkworm (Bombyx mori L.), rust poses a significant threat to the sericulture industry because the disease can decrease production and quality of mulberry foliage.

First Report of Downy Mildew caused by Pseudoperonospora cannabina on Cannabis sativa in New York.

Hemp (Cannabis sativa <0.3% tetrahydrocannabinol) is an emerging crop used for grain, fiber, and cannabinoid production (Fike et al. 2020). In New York, hemp is grown both in controlled environment facilities, including greenhouses, and as a field crop. In August 2020, downy mildew-like symptoms were observed on leaves and inflorescence of hemp plants in a field research trial in Ithaca, NY. Several cultivars, including 'Auto CBD', were affected. Disease was severe with some plants reaching 75% disease severity at the individual plant level. In the most severely affected plots, there was no marketable yield. The disease was characterized by chlorotic and necrotic lesions producing sporangiophores under high humidity. Pigmented sporangia were produced on branched sporangiophores. On artificially inoculated leaves incubated at 18°C, 80% humidity, 12h light for 5d, sporangiophores produced 8-19 pigmented, lemon-shaped sporangia with mean ± SD dimensions of 25.2 ± 3.0 (18.9 to 30.4) x 18.2 ± 2.1 (14.6 to 23.2) µm (n=50). Each sporangium produced 2-5 zoospores after less than 45 min in water at room temperature (22°C). Sporangia were collected from sporulating lesions and DNA was extracted as outlined in Crowell et al. (2020). Fragments of the ribosomal internal transcribed spacer (ITS) region (White et al. 1990), the beta-tubulin ras-associated ypt1 gene (Moorman et al. 2002), and the mitochondrial cytochrome B oxidase subunit 2 (cox2) gene (Hudspeth et al. 2000) were amplified by PCR and sequenced bidirectionally. Sequences were deposited in GenBank under accession numbers OK086084, OM867581, and OM867580, respectively. BLAST searches using the amplified ITS and cox2 sequences resulted in 100% identity to Pseudoperonospora cannabina (HM636051.1, HM636003.1) with ypt1 aligning at 97.95% identity (382/390 bp) with P. cannabina (KJ651402.1). The molecular characterization identified the causal agent as P. cannabina. A representative isolate was deposited in the Cornell Plant Pathology Herbarium as CUP-070922. Sporangia were rinsed from detached leaves and used to confirm pathogenicity on whole plants. Ten 4-week-old 'Anka' plants were spray-inoculated until run off with a suspension of 1x104 sporangia mL-1. Ten control plants were sprayed with water. After inoculation, plants were placed in a 19˚C growth chamber with a 12-h photoperiod and misted for 30 min twice daily to maintain humidity above 80%. Sporangia and previously described symptoms were observed 7 days post-inoculation, while control plants were asymptomatic. The pathogen was reisolated onto detached leaves of 'Anka' from inoculated leaves where both sporangia and oospores were observed. The reisolated pathogen was confirmed morphologically and molecularly, through PCR amplification and bidirectional sequencing of the ITS, cox2, and ypt1 genes, as P. cannabina. To our knowledge, this is the first report of P. cannabina causing hemp downy mildew in New York. Depending on the severity and timing of infections, this disease could pose a significant threat to hemp production in the state. Other members of the genus, P. cubensis and P. humuli cause downy mildew on cucurbits and hops, respectively. As these can cause devastating diseases on their hosts, P. cannabina must be monitored with vigilance as an emerging pathogen (Purayannur et al. 2021; Savory et al. 2011). Literature Cited: Crowell, C. R., et al.2020. Plant Dis. 104:2949. DOI 10.1094/PDIS-04-20-0718-RE Fike, J. H., et al. 2020. Page 89 In: Sustainable Agriculture Reviews, vol 42. Springer, Cham, Switzerland. DOI 10.1007/978-3-030-41384-2_3 Hudspeth, D. S. S., et al. 2000. Mycologia 92:674. DOI 10.2307/3761425 Moorman, G. W., et al. 2002. Plant Dis. 86:1227. DOI 10.1094/PDIS.2002.86.11.1227 Purayannur, S., et al. 2021. Mol. Plant Pathol. 22:755. DOI 10.1111/mpp.13063 Savory, E. A., et al. 2011. Mol. Plant Pathol. 12:217. DOI 10.1111/j.1364-3703.2010.00670.x White, T. J., et al. 1990. Page 315 In: PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego, CA. DOI 10.1016/B978-0-12-372180-8.50042-1.

First Report of Watermelon Mosaic Virus Naturally Infecting Coriander (Coriandrum sativum) and causing a leaf mottling disease in California.

Watermelon mosaic virus (WMV, genus Potyvirus, family Potyviridae) is a species of considerable economic importance to cucurbit crops worldwide (Keinath et al. 2017). This virus has a wide host range that includes more than 170 plant species from 27 families (Dong et al. 2017; Lecoq et al. 2011). In 2018, leaves of coriander (Coriandrum sativum) plants in a student garden (C-SG) at UC Davis, and in a home garden in Davis, CA (C-Pet) (~1.1 miles apart) showed symptoms of light green mottling and crumpling. Symptomatic leaves from each location were weakly positive with the general potyvirus immunostrip test (Agdia, Elkhart, IN). In RT-PCR tests with total RNA extracts (RNeasy Plant Mini Kit Qiagen, Germantown, MD) of these leaves and the potyvirus degenerate primer pair CIFor/CIRev (Ha et al. 2008), the expected-size ~0.7 kb fragment was amplified. These fragments were gel-purified and sequenced, and a BLASTn search revealed highest identities of 91.6% (C-SG) and 97.9% (C-Pet) with the sequence of an isolate of WMV from watermelon in the U.S. (TX29, KU246036). Thus, these isolates are designated WMV-C-SG-18 and WMV-C-Pet-18. Mechanical inoculation experiments were next performed with sap prepared with symptomatic coriander leaf tissue in ice-cold 0.01 M phosphate buffer (pH 7.0) in a 1:4 wt/vol ratio. First, to obtain pure isolates, sap was inoculated onto celite-dusted leaves of Chenopodium quinoa plants (3-4 leaf stage). As expected for WMV, leaves inoculated with sap of each isolate developed chlorotic local lesions ~9 d post-inoculation (dpi) (Moreno et al. 2004). One lesion for each isolate was excised, ground in phosphate buffer, and the sap was mechanically inoculated onto leaves of Nicotiana benthamiana plants. By ~14 dpi, newly emerged leaves showed mild mottling and crumpling, and were weakly positive with the potyvirus immunostrip test. To confirm that these plants were only infected with WMV, total RNA was extracted from symptomatic leaves and used for high throughput sequencing (HTS) (Soltani et al. 2021) at the Foundation Plant Services at UC Davis. The HTS analyses revealed infection with only WMV, i.e., no other viral contigs were identified, and allowed for determination of the complete sequences (~10,000 nt) of WMV [US-CA-C-SG-18] and WMV [US-CA-C-Pet-18] with GenBank accession numbers: OM746964 and OM746965, respectively. Whole genome sequence comparisons revealed that the sequences are 99.0% identical, and 97.3% identical to the sequence of WMV TX29. Sap from symptomatic N. benthamiana leaves infected with each isolate was mechanical inoculated onto leaves of coriander plants (30-35 d old). Newly emerged leaves developed epinasty, crumpling and light green mottling by 14 dpi, and WMV infection was confirmed by RT-PCR with the WMV-specific primer pair WMV-UNI-1F and WMV-UNI-1R (Kim et al. 2019). Thus, Koch's postulates were fulfilled for this leaf mottling disease of coriander. Furthermore, the isolates from coriander induced stunting and distortion and mosaic in leaves of melon, pumpkin and squash plants by 7 dpi, whereas watermelon plants developed stunting and small leaves with mild mottling by 20 dpi. Similar results were obtained with sap prepared from infected coriander leaves. Thus, infected coriander plants are a potential inoculum source for cucurbits via several aphid vectors (Keinath et al. 2017). This is the first report of a mottle disease of coriander caused by WMV, and adds to the wide host range of the virus.

Measuring resistance in durum wheat-Zymoseptoria tritici interaction using aggressiveness quantitative traits

Phenotyping for quantitative resistance to Septoria tritici blotch (STB), one of the most economically devastating diseases of durum wheat (Triticum durum Desf.) in North African region, is a limiting factor for breeding progress. Here we tested main phenotypic markers of aggressiveness affecting host damage and pathogen reproduction reported in earlier studies for measuring differences in quantitative host resistance. A collection of 11 Tunisian durum wheat genotypes varying in resistance to the pathogen was inoculated by a highly aggressive and virulent isolate of Zymoseptoria tritici in a replicated field and controlled experiments, conducted in 2017-2018 at the experimental station of the Regional Field Crops Research Center of Beja in Tunisia. The severity of infection caused by Z. tritici isolate, including percentage of leaf area covered by chlorotic lesions (PLACCL), percentage of leaf area covered by necrosis (PLACN), and percentage of leaf area covered by pycnidia (PLACP) as well as latent period (LP) and pycnidia density (PYCdens) were assessed visually and quantified. The results showed that there were significant differences in all assessed traits on the tested genotypes and significant positive correlations were obtained between PLACN, PLACP and PYCdens. Similarly, there was a significant negative correlation between LP and these last traits. The PLACP was found as the more reliable trait to discriminate genotypes to different classes of resistance, consistent with the results obtained for the remaining traits. These data suggest that using both necrotic leaf area (PLACN) bearing pycnidia and PYCdens can improve selection for quantitative STB resistance.

Molecular characterization of a novel gammacarmovirus infecting cucurbits in India

In this study, we describe the identification of a new gammacarmovirus infecting Cucurbita pepo plants showing a range of mosaic, stunting, yellowing, and wilting symptoms. The virus had a narrow host range and mostly produced chlorotic and necrotic local lesions in the majority of the tested plants. However, Nicotiana benthamiana showed systemic symptoms under laboratory conditions. Using a combination of Sanger sequencing and rapid amplification of cDNA ends (RACE), the complete genome sequence of the virus was determined to be 4274 nucleotides (nt) in length. Its genome organization is similar to that of members of the genus Gammacarmovirus in the family Tombusviridae, consisting of five overlapping open reading frames (ORFs) encoding p28, replicase, p7A, p7B, and coat protein (CP), respectively. The genome is flanked by short 5' and 3' non-coding regions (NCR) at either end. In pairwise comparisons of replicase and CP sequences, the virus showed the highest amino acid sequence identity of 71.55% and 54.86%, respectively, to melon necrotic spot virus (MNSV), the type member of the genus Gammacarmovirus. Since the sequence identity values are below the species demarcation threshold suggested by the International Committee on Taxonomy of Viruses (ICTV), the virus from Cucurbita pepo plants, for which the name "cucurbit carmovirus" (CuCV) is proposed, represents a new species. In phylogenetic analysis based on the replicase and CP amino acid sequences, CuCV clustered with MNSV but formed a distinct branch, further confirming that the virus is a distinct member of the genus Gammacarmovirus.

Cladosporium cladosporioides causes leaf blight on garden pea in Telangana, India.

Garden pea (Pisum sativum L.) is an important vegetable and pulse crop grown worldwide. Leaf blight symptoms with chlorotic lesions were observed on the pea genotypes, 2006-2008A and Arkel in field conditions at Asian Vegetable Research and Development Centre (AVRDC), Hyderabad (17°23' N; 78°29' E) and greenhouse conditions at University of Hyderabad, Hyderabad, India (17° 27' N; 78° 19' E) since 2010. The blight symptoms showed black lesions coated by a fine velvety layer of olive-grey conidia. The disease incidence on field and greenhouse pea plants ranged from 3-6%. The Cladosporium isolates, Gp01 and Gp02 were isolated from pea genotypes, Arkel and 2006-2008A in greenhouse and field conditions respectively. One fungus was predominantly isolated from infected leaf tissue plated onto potato dextrose agar (PDA), and it was identified as Cladosporium sp. based on colony morphology and conidial appearance. Single-spored cultures grown at 26±2 °C on PDA showed olive green mycelia on the obverse-side and dark green on reverse-side (Fig. 1 a,b). The conidia were limoniform with dimensions ranging from 5.91-7.33 x 3.19-4.58 µm (Table 1) and were produced on solitary conidiophores containing 1-2 septa, with no constriction whereas the aerial hyphae were septate and unbranched or rarely branched (Fig.1 c-d). To verify the pathogenicity of the fungus, detached leaf assay was carried on 30-day old Arkel leaves by point inoculation with 20 µL (106 spores/ml) spore suspension using a needle-less syringe to infiltrate the spores into the abaxial surface and the inoculated leaves were incubated at 26±2 °C for 3 days in petri plates containing moistened autoclaved absorbent cotton. Pea leaves inoculated with water served as controls. The experiment was repeated with a minimum of three times with five leaves per replication and three replications per experiment for each of the isolates. The appressorial germ tube formation was observed by scanning electron microscopy (SEM) at 12 hours post inoculation (hpi) (Fig.1 e-f). Thirty-day old pea plants were spray-inoculated with a spore suspension of 1-2 ml per plant with a concentration of 106 spores/ ml while the control plants were mock-inoculated (sprayed) with water. 5 plants per replicate per isolate with three replications per experiment and a minimum of three experiments were carried. The inoculated plants were covered with polythene zip-lock bags for two days post inoculation (dpi) and maintained in greenhouse at 26±2 °C with natural photoperiod of 14 hours. Wilting and necrosis symptoms accompanied by chlorotic lesions were observed on pea plants at 7 days post inoculation (dpi) (Fig. 2). Complying with Koch's postulates, the pathogen, which was re-isolated from the first appearance at 3 dpi from infected pea showed similar morphological characteristics as those used for inoculation. Sequencing analysis of the internal transcribed spacer (ITS) region using ITS1 and ITS4 primers (White et al. 1990), D1-D2 region sequencing (Kwiatkowski et al. 2012), Translation elongation factor 1-alpha (TEF) and Actin (ACT) gene sequencing (Nam et al. 2015) of Gp01 and Gp02 showed significant similarity with C. cladosporioides (Table S1). Further, concatenated phylogenetic trees constructed by maximum likelihood method using all the four (ITS, D1-D2, TEF and ACTIN) gene sequences of these isolates along with other reported Cladosporium species (Fig. S1a) and specific isolates of C. cladosporiodes which are reported as "sensu stricto" (Bensch et al. 2010) collected from different substrata (Fig. S1b) confirmed them as C. cladosporioides. Gp01 and Gp02 were also confirmed by Microbial Type Culture Collection (MTCC, India) as C. cladosporioides with Acc. No. MTCC 9994 and MTCC 9995. C. cladosporioides has been previously reported on Dalbergia sp. and Eucalyptus sp in India and on garden pea in other countries (Bensch et al. 2010). However, owing to the impact of this pathogen on growth and productivity of pea crop, further in-depth study needs to be carried. To our understanding this is the first report of Cladosporium cladosporioides infecting garden pea in India.

Black rot disease incited by Indian race 1 of Xanthomonas campestris pv. campestris in Brassica juncea L. cv. Pusa Bold in India.

Mustard (Brassica juncea L.) is an important oil seed crop in the Brassicaceae family. It is widely cultivated in India for its edible leaves, oil and medicinal properties. In January 2022, we noticed necrotic symptoms typical black rot disease on Brassica juncea (L.) cv. Pusa Bold grown in Indian Agricultural Research Institute, India. Initially, chlorotic lesions emerged on the leaf margin, which progressed to angular V-shaped necrotic lesions and blackened veins. Disease progression became a necrotic appearance in the leaf results browning and papery leaf texture appeared. The suspected causal agent was isolated from three different diseased plants of Pusa Bold on nutrient sucrose agar medium that formed pale yellow, mucoid, and fluidal colonies. Three representative isolates originated from three different plants were sub-cultured on YGCA medium. These isolates are Gram-negative, oxidase negative, KOH positive, nonfluorescent on King's Medium B agar, and positive for starch hydrolysis test (Schaad and White 1974). The 16S ribosomal RNA gene and avirulence genes - AvrBs1 and AvrGf1 were amplified and sequenced in these three isolates with other Xanthomonas campestris pv. campestris (Xcc) isolates. The DNA sequence analysis revealed that these isolates are within the species of X. campestris. The race 1 specific marker namely xcc-b100_4389 was used to characterized the race by detection of 1090bp fragment respectively from gDNA of Xcc isolates (Rubel et al., 2017). The pathogenicity of these isolates was tested twice on youngest leaves of 30-day-old plants of Pusa Bold to convey Koch postulates. Inoculum of three isolates were prepared in nutrient broth at 28°C for 48-h. The pathogenicity test was conducted by small scissors dipped in a bacterial suspension (~ 108 cfu/ml) to cut leaf near margins at 10 points per leaf and the three youngest leaves per plant with three replications. The number of infected points per leaf and the severity of symptoms were assessed 15 and 30 days after inoculation (Singh et al., 2011; 2016). The chlorotic lesions with V-shaped symptoms were appeared on all inoculated plants after 15 and 30 dpi (days post-inoculation). The bacteria were reisolated from inoculated plants and has the same identity as original isolates by using 16S rRNA, avr genes and race 1 specific marker PCR, thereby confirming Koch's postulates. The bacterial inoculation was repeated and the same symptoms appear. Most of the crucifers are infected with black rot disease e.g., cauliflower, cabbage, Brussels, sprout etc. (Vicente et al., 2001). The nucleotide BLAST analysis of 16S rRNA, AvrBs1, AvrGf1 showed a 100% identity with different Xcc strains reported from Germany (B100; AM920689), Brazil (ATCC 33913; AE008922), India (Xcc-C7; CP077958), France (CFBP 5817; CM002673) and China (8004; CP000050) (Singh et al. 2022). Whilst, the nBLAST analysis of xcc-b100_4389 showed 100% nucleotide identity with Xcc race 1 (B100; AM920689), Germany. The sequences were deposited in GenBank (16S rRNA: OM839780; AvrBs1: OM994397; AvrGf1: OM994398; xcc-b100_4389: OM994399). The XccAK1 strain (ITCCBH_0014) was deposited in Indian Type Culture Collection, ICAR-IARI, New Delhi, India. Presently, it is a first report of necrotic black rot on B. juncea cv. Pusa Bold incited by Xcc race 1, India. Previous research reported the black rot disease on other species of the Brassica genus e.g., B. oleracea, and B. napus in Serbia (Popovic et al., 2013) and Argentina (Gaetan et al., 2005).

First report of sunflower chlorotic mottle virus infecting sunflower plants in Brazil

Sunflower (Helianthus annuus) plants showing symptoms of chlorosis, mosaic, chlorotic ringspot, and necrosis on younger leaves were found in a small experimental plot in Piracicaba, in the state of São Paulo, Brazil. Preliminary examinations by transmission electron microscopy of symptomatic leaf tissue revealed flexuous filamentous particles 13-15 nm wide and 700-750 nm long, and cytoplasmatic cylindrical inclusions typical of those found in plant cells infected by members of the Potyvirus genus. Total RNA extracted from symptomatic leaves and subjected to RT-PCR followed by partial nucleotide sequencing confirmed the presence of a potyvirus in the affected plants, which was identified as sunflower chlorotic mottle virus (SuCMoV), a member of the Sunflower chlorotic mottle virus (genus Potyvirus, family Potyviridae) species. Mechanical transmission assays with extracts of symptomatic sunflower leaves reproduced the original symptoms in sunflowers, mosaic symptoms in Zinnia elegans, and chlorotic local lesions in Chenopodium amaranticolor and C. quinoa. Sunflower and zinnia plants became infected after aphid transmission experiments with Myzus persicae. RT-PCR tests using specific primers for SuCMoV confirmed the presence of this virus in experimentally infected plants, meeting the criteria of Koch’s postulate. This is the first report of SuCMoV infecting sunflower plants in Brazil.

First Report of Alternaria tenuissima Causing Leaf spot on Luffa cylindrica in China.

In August 2020-2021, symptoms of leaf spot were observed in luffa (Luffa cylindrical) fields in Qingdao city, Shandong Province. In all the 10 fields investigated, leaf spot occurred. The incidence (% luffa plants with symptoms from a defined number of plants assessed) was 35 to 60%. Early symptoms of infected leaves were small and irregular chlorotic lesions which later became irregular brown spots. As the disease progressed, the lesions gradually spread from the edge to the center of leaves to the middle, and became dark brown. The enlarged spots coalesced and eventually led to the withering and death of the leaves. In order to isolate the pathogen, 30 symptomatic leaves were collected from different planting fields. Small pieces of leaf tissues (5×5 mm) were cut from the junction of healthy and diseased tissues, sanitized with 2% NaClO for 1 min, rinsed three times with sterile distilled water. The tissue samples were then placed on potato dextrose agar (PDA) amended with 50 mg/L streptomycin sulfate, and incubated at 28℃ for 5 days in the dark. Ten purified fungal isolates were obtained by single spore isolation method. Colonies of these fungal isolates on the PDA medium were initially grayish-white, and then turned olive green with abundant cotton-like aerial hyphae. On potato carrot agar (PCA) medium, these fungi produced light brown and solitary conidiophore with septum. Conidia were obclavate or ellipsoid, brown, with 1-5 transverse septa and 0-3 longitudinal septa, and measured 13.2 to 49.5 × 9.5 to 21.6 µm (n=50). The morphological characteristics of these isolates were consistent with that of Alternaria spp. (Simmons 2007). The representative isolate NEAU-SG-1 was selected for molecular identification. The internal transcribed spacer (ITS) region of ribosomal DNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-α gene (TEF), histone 3 (HIS3), and RNA polymerase II second largest subunit (RPB2) were amplified using primer pairs ITS1/ITS4 (White et al. 1990), gpd1/gpd2 (Berbee et al. 1999), EF1-728F/EF1-986R (Carbone and Kohn 1999), H3-1a/H3-1b (Glass and Donaldson 1995), and RPB2-5F2/fRPB2-7cR (Sung et al. 2007), respectively. Sequences of these genes of isolate NEAU-SG-1 were deposited into GenBank database with the accession numbers of OL307719, OL415166, OL415169, OL415167, and OL415168. BLAST analysis of these sequences showed 99-100% homology with sequence homology with Alternaria tenuissima strains (ITS, MH824269; GAPDH, MK683783; TEF, MN056178; HIS3, MH824371; RPB2, LC621694). To fulfill Koch's postulates, ten surface disinfected 30-day-old luffa seedlings were inoculated by spraying conidia suspension (106 conidia/ml) of isolate NEAU-SG-1. The other ten surface disinfected seedlings inoculated with sterile distilled water served as the control group. After inoculation, each plant was covered with plastic bags for three days and cultured in greenhouse at 25℃. One week later, leaves inoculated with conidia suspension were observed with the same symptoms as described above, while the leaves of the control group were asymptomatic. Pathogenicity test was repeated twice. The Alternaria isolates were successfully re-isolated from those infected leaves and identified using the morphological and molecular methods described above. A. tenuissima has a wide host range in the world, and is the pathogen of leaf spot of many crops (Ma et al. 2021). To our knowledge, this is the first report of A. tenuissima causing leaf spot on luffa in China. This report will provide basic information for the diagnosis and prevention and control strategies of luffa leaf spot.

Exploring the Host Range of Rose rosette Virus among Herbaceous Annual Plants.

To study the host range of Rose rosette virus (RRV), we employed crude sap inoculum extracted from RRV-infected roses and the RRV infectious clone. We inoculated plants from the families Solanaceae, Cucurbitaceae, Leguminosae, Malvaceae, Amaranthaceae, and Brassicaceae. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect RRV in the inoculated plants throughout their growth stages. Interestingly, RRV was detected in the newly developed leaves of tomato, pepper, tobacco, cucumber, squash, zucchini, pumpkin, pea, peanut, soybean, spinach, okra, and Chenopodium spp. The speed of upward advancement of RRV within infected plants was variable between plants as it took two to three weeks for some plant species and up to five weeks in other plant species to emerge in the newest leaves. No severe symptoms were detected on most of the inoculated plants. Chenopodium spp., spinach, cucumber and Nicotiana rustica exhibited either chlorotic or necrotic lesions with variable shapes and patterns on the systemically infected leaves. Double membrane-bound particles of 80-120 nm in diameter were detected by transmission electron microscopy in the infected tissues of cucumber, pepper, and N. benthamiana plants. This finding infers the validity of mechanical inoculation for RRV on a wide range of plants that would serve as potential natural reservoirs.

PM 7/153 (1) Mechanical inoculation of test plants

PM 7/153 (1) Mechanical inoculation of test plants

Molecular and biological characterization of an isolate of the potyvirus passiflora virus Y naturally infecting soybean (Glycine max) in Brazil.

Passiflora virus Y was detected naturally infecting soybean (Glycine max) for the first time in Brazil. Here, we report the nearly complete genome sequence and molecular and biological properties of the PaVY-Br isolate. The nearly complete genome sequence is 9679 nt long and shares 84.4% nt sequence identity with a previously reported PaVY isolate from Passiflora sp. PaVY-Br induced chlorotic spots and systemic mosaic on soybean and chlorotic local lesions on yellow passion fruit (Passiflora edulis) and sesame (Sesamum indicum). The virus was successfully transmitted by Myzus persicae, indicating that this aphid vector can contribute to the spread of PaYV from passion fruit to soybean plants. Additional epidemiological research is in progress to investigate the distribution of PaVY in soybean production areas in Brazil.

First Report of Tar Spot on Corn Caused by Phyllachora maydis in Georgia, United States.

Tar spot is a major foliage disease of corn (Zea mays) caused by the fungus Phyllachora maydis. This disease can reduce the quality of silage, stover, husks, and grain (Valle-Torells et al. 2020). It was identified in the United States for the first time in 2015 in northern Illinois and Indiana (Ruhl et al. 2016). As of 2020, it has also been confirmed in Iowa, Michigan, Wisconsin, Ohio, Florida, Missouri, Minnesota and Pennsylvania (Telenko et al. 2020, Collins et al. 2021). In early August 2021, tar spot symptoms and signs were observed in late-planted, hybrid corn in southwestern Georgia in both Tift and Calhoun Counties. Where it occurred, incidence was nearly 100% and severity ranged from approximately 1% to 20% leaf coverage. As of November 2021, Tar spot has been confirmed in 13 counties throughout South Georgia. The symptoms on leaves were chlorotic lesions and the signs were black, raised, circular to irregular-shaped structures (stromata or clypei) ranging from 0.2 mm to 8 mm on the surface of the leaves. The stromata were present on both green and necrotic leaf tissue. Southern corn rust (Puccinia polysora) was often, but not always observed on leaves with tar spot. Microscopic observations of stromata included single and clustered ascomata (60 × magnification) that contained cylindrical, unitunicate asci with ellipsoid, hyaline, aseptate ascospores and abundant filiform paraphyses (400 × magnification). For molecular confirmation, stromata were surface sterilized with 95% ethanol and separated from the leaf tissue, then DNA was extracted using the Qiagen DNeasy Plant Mini-Kit (Qiagen, Inc., Valencia, CA). PCR was conducted with the universal ITS1 and ITS4 primers to amplify the internal transcribed spacer (ITS) region of the rDNA. The amplicons were Sanger sequenced (Genewiz, Inc., South Plainfield, NJ) and a consensus sequence of a representative lesion was deposited in GenBank (accession number OK649959). Using the BLASTN algorithm our sequence shared 100% and 99.2% coverage, and 97.2% and 98.4% similarity to P. maydis GenBank accessions MG881848 and MK184990, respectively. Phyllachora maydis is an obligate parasite that cannot be cultured, so Koch's postulates were not performed. Inoculum of P. maydis may have been transported to Georgia from Florida by wind or other weather events, but this has not been confirmed.

First Report of Tomato Fruit Blotch Virus Infecting Tomatoes in Brazil.

Recently, a new blunervirus was reported in tomatoes showing fruit chlorotic lesions. This virus, named tomato fruit blotch virus (ToFBV), was found associated with the tomato fruit blotch disease in Italy and Australia, even though Koch's postulates were not fulfilled and no viral particles were seen in leaf dips observed with an electron microscope (Ciuffo et al. 2020). In December 2019, symptoms of circular or irregular chlorotic blotches were observed in tomato fruits in an organic farm in Distrito Federal, Brazil. Five different tomato cultivars (2100 plants of cv. Sweet grape, 1700 of Giacomo, 560 of Grazianni, 160 of Tropical, and 160 of DRC 5640) were being grown in two greenhouses and all of them presented the symptoms in at least one fruit, particularly in older fruits. No virus-like symptoms were observed in young and middle leaves, but older leaves could not be examined because they were removed as a routine activity of the farm; and also due to the moderate infestation of the tomato russet mite Aculops lycopersici, associated with leaf and stem necrosis. No viral particles were observed in an electron microscope analysis of symptomatic fruit tissues, and sap inoculation and grafting of stems did not produce any symptom in indicator plants. Two young and asymptomatic plants with the first fruits still in development were removed from another greenhouse of the farm and transported to our greenhouse, but the typical blotch symptoms neither appeared in the fruits nor the necrosis symptoms in the leaves. Serological tests performed for all collected leaf and fruit samples using antibodies produced in-house against common tomato-infecting tospoviruses and potyviruses were negative, as well as a polymerase chain reaction (PCR) detection test for begomoviruses (Rojas et al. 1993). Total RNA from newly collected samples consisting of one symptomatic fruit sample and five asymptomatic leaf samples from distinct plants were individually extracted using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and pooled for next generation sequencing (NGS). The library was constructed using TruSeq Stranded Total RNA with Ribo-Zero Plant (Illumina, San Diego, USA) and sequenced at Macrogen, Inc. (Seoul, South Korea) in an Illumina Novaseq6000 platform. The 4,621,977,958 reads obtained were trimmed using Trimommatic 0.35 (Bolger et al. 2014) and contigs were assembled using Velvet (Zerbino and Birney 2008). Following tblastx analysis on Geneious 9.1.8 (Biomatters Ltd.) and BLAST on the NCBI platform (Altschul et al. 1990), seven contigs matching tomato chlorosis virus (ToCV) and five contigs matching ToFBV were identified. Sequences for each of the four genome components of ToFBV (MK517477-MK517480) already present in databases were used as reference using the Map to Reference function in Geneious. A total of 338,402, 78,039, 555,302 and 461,474 reads mapped to virus genome components 1 to 4, respectively, with >99% coverage for each. Four final consensus sequences were used for BLAST analyses on NCBI and presented 97 to 99.7 % nucleotide identity with those used for mapping. These sequences were deposited in GenBank as isolate MAL under accession numbers MW546267 (RNA 1, 5770 nt), MW546268 (RNA 2, 3612 nt), MW546269 (RNA 3, 2826 nt) and MW546270 (RNA 4, 1950 nt). The primer pair Bluner1F (5'-ATTCCTGTTCCTTCGGATAAACTCGT-3') and Bluner1R (5'-CACACGTGCAGGAAATGGAAAGA-3') directed to RNA 1 was used to specifically detect the virus. Three leaf samples and two fruit samples, each from a different plant with typical symptoms, were tested positive for ToFBV and negative using ToCV-specific primers in RT-PCR (Dovas et al. 2002). This confirmed that although some plants pooled in the HTS library were infected with ToCV, the chlorotic blotch symptom was clearly associated with the presence of ToFBV. Furthermore, the ~0.5 kbp amplicon for ToFBV-specific primers from one randomly selected sample was sequenced with both primers and the resulting sequence shared 100% nt identity with the RNA 1 of ToFBV isolate Fondi2018 from Italy (MK517477). Then, the virus was detected in the tissue from the surface of another fruit, but not from its internal part, suggesting a superficial infection. The findings presented here are of high phytosanitary significance, given the strong symptoms associated with tomato fruit blotch disease and the identification of ToFBV in the tomato samples from Brazil.

Identification and Characterization of Colletotrichum fructicola and Colletotrichum siamense Causing Anthracnose on Luffa Sponge Gourd in China.

Luffa sponge gourd (Luffa cylindrica) is an important cucurbitaceous vegetable and is known as the source of loofah. From 2020 to 2021, a leaf disease occurred on luffa leaves in the Hunan Province of China. Symptoms were displayed as oval to irregular chlorotic lesions surrounded by yellow halos. The pathogens were isolated from the affected leaves. According to morphological characterization and molecular identification using multi-locus phylogenetic analysis of the internal transcribed spacer (ITS), actin (ACT), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin (TUB2), and partial mating type (Mat1-2) gene (ApMAT) regions, the pathogens were identified as two Colletotrichum species: Colletotrichum fructicola and C. siamense. Koch’s postulates were identified by a pathogenicity test and re-confirmation. To the best of our knowledge, C. fructicola and C. siamense are two new species associated with luffa sponge gourd anthracnose.

First Report of Schlumbergera Virus X in Dragon Fruit (Hylocereus spp.) in Spain.

Dragon fruit (Hylocereus undatus) is a high-value fruit crop, introduced about a decade ago in the mainland of Spain. In 2021, chlorotic spots were observed on young cladodes in a commercial dragon fruit orchard in the province of Seville (southern Spain). Sap extracts from 4 symptomatic cladodes were used to mechanically inoculate indicator plants: no symptoms were produced in Datura stramonium plants, but Chenopodium amaranticolor reacted with chlorotic local lesions and prickly pear plants (Opuntia ficus-indica) showed irregular yellow ringspot symptoms on young cladodes at 30 days post inoculation. Total RNA was extracted from all 4 symptomatic cladodes as previously described (Pallas et al. 1987). Reverse transcription (RT)-PCR, which was carried out with M-MLV-RT and Go Taq Pol (Promega Biotech Ibérica, SL, Madrid, Spain) and tobamovirus primers (Dovas et al. 2004), failed to produce any amplicons. Electrophoretic analysis of dsRNA, extracted from symptomatic cladodes, yielded a banding pattern similar to the one reported for potexviruses (Valverde et al. 1986). Primers specific for Cactus virus X (Kim et al., 2016) failed to produce amplicons, whereas potexvirus group primers (Potex F5/Potex R2) (van der Vlugt and Berendsen 2002), amplified an expected 584-bp amplicon from RNA extracts of all 4 field-collected samples. The RT-PCR products from the four samples were Sanger-sequenced. All showed identical sequence results (GenBank Accession MZ614940) with a predicted amino acid identity of 99% with the corresponding RNA-dependent RNA polymerase amino acid sequence of Schlumbergera virus X (SchVX) (GenBank Accession No. ACD99908). SchVX-specific primers (431s, 5'-TTTGAGGAGTTCGTCAGCAAGA-3' and 431As, 5'-TCAAGAGCCCATTGAGAGAGTG-3') that were designed based on the new sequence, amplified the expected amplicon of around 430 nucleotides from the total RNA extracts of the four samples. The amplicons were Sanger-sequenced and the expected nucleotide sequence was obtained. This pair of primers were used in RT-PCR tests on subsequent surveys in 2 commercial dragon fruit greenhouses from the province of Seville, and in 1 experimental greenhouse in the province of Almeria. All samples from 25 symptomatic plants of H. undatus, H. hybridum, H. costaricensis, and H. purpusii in Seville and from 1 symptomatic H. undatus plant from Almeria tested positive for SchVX, while 15 asymptomatic plants tested negative. The results obtained in this investigation support that SchVX is present in the cladodes of dragon fruit plants expressing the symptoms. SchVX has been reported previously from H. undatus from Brasil (Duarte et al. 2008) and from prickly pear in Mexico (De La Torre-Almaráz et al. 2016), and to our knowledge, this is the first report of the virus in Spain. These findings suggest that SchVX has been introduced in dragon fruit farms from Spain and propagation of this emerging crop through planting of cuttings should include testing for this virus in order to prevent further spread.

Identification of Resistance Sources against Bacterial Pustule Disease Caused by Xanthomonas axonopodis pv. glycines in Soybean using Excised Leaf Technique

Bacterial pustule of soybean is a foliar disease caused by Xanthomonas axonopodis pv. glycines. The organism produces pustules which causes premature defoliation and ultimately results in significant reduction in seed yield and quality. Use of resistant cultivars can be a cost effective and eco-friendly approach to address the issue. This study was aimed at development of a rapid and reliable in-vitro screening method and screening of soybean genotypes for the identification of resistance sources to bacterial pustule disease. One hundred thirty-six soybean genotypes including released varieties, indigenous and exotic germplasm were screened by excised leaf technique for their reaction to bacterial pustule disease. An improved in-vitro technique for screening of bacterial pustule disease was developed. Rooting and survival studies indicated that soybean leaves excised from 25 days old plants could survive up to 30 days at room temperature in plain water. Inoculation with bacterial suspension of 107 to 109 colony forming units (cfu/ml) at 27±1°C under the photoperiod of 12h/day light showed initiation of chlorotic lesions 48 hours after inoculation. Based on the latent period and appearance of the chlorotic lesions, the genotypes were classified as susceptible, moderately resistant and resistant. Four soybean genotypes (TS-3, P-4-2, Hara Soya and Himso 1685) were resistant while, seventeen genotypes (AMS MB5-19, Bhatt, DSb-12, JS-335, JS93-05, MACS- 1188, MAUS- 71, PK-262, PS- 1241, Pusa-5, RKS- 18, SL-688, SL-744, SL-958, SL97-52, SL-979, and T99-76) were moderately resistant and rest of the genotypes were susceptible to bacterial pustule disease.

First Report of Molecular Detection of Leveillula taurica Associated with Powdery Mildew of Linseed (Linum usitatissimum) from India.

Linseed (Linum usitatissimum L.) is an important oil yielding crop cultivated widely in India. During the field survey, 2018-19 (Rabi season; January-February), characteristic powdery mildew disease symptoms were observed in around 90 hectares of linseed in Northern Karnataka (Agricultural Research Station, Raichur). The incidence of the disease was estimated, and it was found in the range up to 96 per cent. Initially, the symptoms were appeared as tan lesions, which later turned into small, circular chlorotic lesions and yellowing of the abaxial leaf surface with white fungal mycelial growth on the abaxial leaf surface. As the disease progress, white fungal mycelial growth was also noticed on the adaxial leaf surface. In severe infection, drying and premature defoliation of infected leaves was observed (Figure S1 A-D). A total of 12 samples were collected and subjected to morphological and molecular identification. Microscopic examination revealed the presence of conidia borne on conidiophores (simple, sometimes branched). Conidiophores were measured 83.17 - 116.45 µm (n=90). Conidia were single-celled, hyaline, dimorphic (pyriform and cylindrical), and 70.2 - 82.7μm × 15.1 - 26.3 μm (n=100) (Figure S1 E&F). A reference specimen was deposited at Agharkar Research Institute's (ARI) Ajrekar Mycological Herbarium (AMH 2015-07/AKC 2231-03/SKS/DKM). Since the conidial morphology and characters were very similar to Leveillula taurica the fungus was identified as L. taurica (Lév.) G. Arnaud. (Braun and Cook, 2012). Further, to confirm the identity based on ITS-rDNA barcoding, genomic DNA was isolated from conidial suspension harvested from powdery mildew affected linseed samples. ITS was amplified using powdery mildew-specific ITS universal primer pair PN23/PN34 and sequenced directly (Chen et al., 2008). The ITS regions were amplified at ~700bp. nBLAST analysis revealed that the ITS sequence shared 100% similarity with reference sequence AB045148.1 respectively. The representative sequences were deposited in GenBank with accession numbers OK036584 (LuLtR1) and OK036585 (LuLtR2). Further amplification of L1/L2 primer pair from ITS region were PCR amplified (Reddy et al., 2013) and an expected amplicon of 374bp was analyzed. Though, the ITS-rDNA and L1/L2 specific region analyzed, they are not specific exclusively to the L. taurica. However, combination of morphology and molecular sequence was used to confirm the identity. Further to prove Koch's postulates, pathogenicity tests were conducted on healthy linseed plants grown under greenhouse condition (28±2° C; >80% Relative Humidity). Conidia were harvested from infected leaves and the suspension was sprayed on 30 (40 days old) linseed plants. The development of powdery mildew symptoms was recorded on 22 plants 10 days after inoculation. Control plants inoculated with sterile water remained healthy, and no powdery mildew symptoms were observed. The identity was confirmed upon microscopic observation of spores from artificially inoculated plants, thus confirming the association. Leveillula taurica is known to cause powdery mildew on tomato, chilli and other crops. Amano (1986) and Saharan and Saharan (1994) reported the occurrence of L. taurica on linseed, however, no further details are available on the association, as well as molecular confirmation of L. taurica associated with linseed from India (Farr and Rossman, 2021). To the best of our knowledge, this is the first report of L. taurica associated with powdery mildew of linseed in India. Since it is the first confirmed record through molecular sequence analysis, it is important to know its geographical distribution and impact on linseed production to chalk the management strategies.

Leaf Blight on Photinia × fraseri Caused by Pestalotiopsis trachicarpicola in China.

Photinia × fraseri is a well-known green plant mainly distributed in the Yangtze River and Yellow River Basin, east and southwest of China (Guan et al. 2013). In October 2020, typical leaf blight symptoms on roughly 10% leaves in a Photinia × fraseri shrub were observed in the campus of Sichuan Agricultural University (30°42'19″ N, 103°51'29″ E). Initially, chlorotic lesions with brown margins occurred on the leaf margin, then the large patches formed to cause leaves necrotic, finally lesions to dry and acervulus bred in 2-4 months later. Five single conidium isolates were carried out (Chomnunti et al. 2014) cultured on potato dextrose agar (PDA) at 25 ℃. All isolates shared similarly morphological characteristics, which was white and thin, and the reverse were yellowish. Mycelium was hyaline, sparsely septate, measuring 1-4 μm in diam. Conidiogenesis formed after 7 days. Conidiogenous cells were discrete, lageniform, smooth, thin-walled, colorless. Conidia were fusiform, straight to slightly curved, 4-septate, 21-30 × 5-7 μm (x ̅= 27 × 6.0 μm, n=30); basal cells were obconic with truncate base, hyaline, thin- and smooth-walled, 4-7 μm long (x ̅= 5.5 μm, n=30); three median cells were doliiform with thick walls, concolorous, olivaceous, constricted at the septa, and septa and periclinal walls were darker than the rest of the cell, 14-20 μm long (x ̅= 17 μm, n=30); apical cells were hyaline, conic to cylindrical, 3.0-6.5 μm long (x ̅= 4.5 μm, n=30), with 2-4 (mostly 3) tubular apical appendages arising from the upper portion, rarely branched, 7.5-18 μm long (x ̅= 12 μm, n=50); basal appendage was single, unbranched, 3-10 μm long (x ̅= 6.5 μm, n=30). DNA was extracted from the representative strain (SICAUCC 21-0012), and the internal transcribed spacer (ITS) region, the large subunit of the nrDNA (LSU), translation elongation factor 1-alpha (tef1-α), and partial sequences of β-tubulin (tub2) were amplified by polymerase chain reaction and sequenced with primers ITS5/ITS4, LR0R/LR5, 728F/1567R, and Bt2a/Bt2b, respectively (Zhang et al. 2012, Ariyawansa & Hyde 2018). The sequences were deposited in GenBank, viz. MZ453106, MZ453108, MZ467300, MZ467301, respectively. The nucleotide blast showed 99% (ITS, 0 gaps), 100% (tub2, 0 gaps), 100% (tef1-α, 0 gaps) identities with the ex-type Pestalotiopsis trachicarpicola Yan M. Zhang & K. D. Hyde (IFRDCC 2440). The fungus was identified as P. trachicarpicola combined with phylogeny and morphology (Maharachchikumbura et al. 2012, Zhang et al. 2012). To conduct Koch's postulates, five healthy 6-year-old P. × fraseri were inoculated with 10 µl spore suspension (106 conidia/ml) onto the wounded sites (five leaves per plant, ~1 to 2 years old) via sterile pin, and five healthy plants treated with sterile dH2O as controls (Yang et al. 2021). The plants were placed in a greenhouse at 25°C with relative humidity >80%. After 2 months, leaf blight symptoms gradually emerged on inoculated leaves, and the controls were symptomless. Fungal isolates from symptomatic plants showed similar morphological characteristics as SICAUCC 21-0012, and the pathogen was not isolated from asymptomatic plants. To our knowledge, this is the first report of leaf blight caused by P. trachicarpicola on Photinia × fraseri in China. Disease management should be adopted properly to restore and improve its ornamental value.