Pub Date : 2024-09-09DOI: 10.1094/PDIS-01-24-0217-SR
Simon P Fraher, Mark Watson, Hoang Nguyen, Savannah Moore, Ramsey S Lewis, Michael Kudenov, G Craig Yencho, Adrienne M Gorny
Meloidogyne spp. (root-knot nematodes [RKNs]) are a major threat to a wide range of agricultural crops worldwide. Breeding crops for RKN resistance is an effective management strategy, yet assaying large numbers of breeding lines requires laborious bioassays that are time-consuming and require experienced researchers. In these bioassays, quantifying nematode eggs through manual counting is considered the current standard for quantifying establishing resistance in plant genotypes. Counting RKN eggs is highly laborious, and even experienced researchers are subject to fatigue or misclassification, leading to potential errors in phenotyping. Here, we present three automated egg counting models that rely on machine learning and image analysis to quantify RKN eggs extracted from tobacco and sweet potato plants. The first method relied on convolutional neural networks trained using annotated images to identify eggs (M. enterolobiiR2 = 0.899, M. incognitaR2 = 0.927, M. javanicaR2 = 0.886), whereas a second contour-based approach used image analysis to identify eggs from their morphological characteristics and did not rely on neural networks (M. enterolobiiR2 = 0.977, M. incognitaR2 = 0.990, M. javanicaR2 = 0.924). A third hybrid model combined these approaches and was able to detect and count eggs nearly as well as human raters (M. enterolobiiR2 = 0.985, M. incognitaR2 = 0.992, M. javanicaR2 = 0.983). These automated counting protocols have the potential to provide significant time and resource savings annually for breeders and nematologists and may be broadly applicable to other nematode species.
{"title":"A Comparison of Three Automated Root-Knot Nematode Egg Counting Approaches Using Machine Learning, Image Analysis, and a Hybrid Model.","authors":"Simon P Fraher, Mark Watson, Hoang Nguyen, Savannah Moore, Ramsey S Lewis, Michael Kudenov, G Craig Yencho, Adrienne M Gorny","doi":"10.1094/PDIS-01-24-0217-SR","DOIUrl":"10.1094/PDIS-01-24-0217-SR","url":null,"abstract":"<p><p><i>Meloidogyne</i> spp. (root-knot nematodes [RKNs]) are a major threat to a wide range of agricultural crops worldwide. Breeding crops for RKN resistance is an effective management strategy, yet assaying large numbers of breeding lines requires laborious bioassays that are time-consuming and require experienced researchers. In these bioassays, quantifying nematode eggs through manual counting is considered the current standard for quantifying establishing resistance in plant genotypes. Counting RKN eggs is highly laborious, and even experienced researchers are subject to fatigue or misclassification, leading to potential errors in phenotyping. Here, we present three automated egg counting models that rely on machine learning and image analysis to quantify RKN eggs extracted from tobacco and sweet potato plants. The first method relied on convolutional neural networks trained using annotated images to identify eggs (<i>M. enterolobii</i> <i>R</i><sup>2</sup> = 0.899, <i>M. incognita</i> <i>R</i><sup>2</sup> = 0.927, <i>M. javanica</i> <i>R</i><sup>2</sup> = 0.886), whereas a second contour-based approach used image analysis to identify eggs from their morphological characteristics and did not rely on neural networks (<i>M. enterolobii</i> <i>R</i><sup>2</sup> = 0.977, <i>M. incognita</i> <i>R</i><sup>2</sup> = 0.990, <i>M. javanica</i> <i>R</i><sup>2</sup> = 0.924). A third hybrid model combined these approaches and was able to detect and count eggs nearly as well as human raters (<i>M. enterolobii</i> <i>R</i><sup>2</sup> = 0.985, <i>M. incognita</i> <i>R</i><sup>2</sup> = 0.992, <i>M. javanica</i> <i>R</i><sup>2</sup> = 0.983). These automated counting protocols have the potential to provide significant time and resource savings annually for breeders and nematologists and may be broadly applicable to other nematode species.</p>","PeriodicalId":20063,"journal":{"name":"Plant disease","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141175552","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Casuarinaequisetifolia is crucial in protecting coastal regions of China against typhoon attacks but has faced a substantial challenge due to wilt disease caused by pathogens of the Ralstonia solanacearum species complex (RSSC). Although the initial outbreak of Casuarina wilt in the 1970s was effectively controlled by disease-resistant C. equisetifolia varieties, the disease has recently re-emerged in coastal regions of Guangdong. In this study, we report the isolation, characterization, and comparative genomic analysis of 11 RSSC strains from diseased C. equisetifolia at various locations along the coast of Guangdong. Phylogenomic analysis showed that the strains were closely related and clustered with phylotype I strains previously isolated from peanuts. Single-gene-based analysis further suggested these strains could be derived from strains present in Guangdong since the 1980s, indicating a historical context to their current pathogenicity. Casuarina-isolated strains exhibited notably higher virulence against C. equisetifolia and peanuts than the representative RSSC strains GMI1000 and EP1, suggesting host-specific adaptations that possibly contributed to the recent outbreak. Comparative genomic analysis among RSSC strains revealed a largely conserved genome structure and high levels of conservation in gene clusters encoding extracellular polysaccharide biosynthesis, secretion systems, and quorum sensing regulatory systems. However, we also found a number of unique genes in the Casuarina-isolated strains that were absent in GMI1000 and EP1, and vice versa, pointing to potential genetic factors underpinning their differential virulence. These unique genes offer promising targets for future functional studies. Overall, our findings provide crucial insights into the RSSC pathogens causing Casuarina wilt in Guangdong, guiding future efforts in disease control and prevention.
{"title":"Pathogenic and Comparative Genomic Analysis of <i>Ralstonia pseudosolanacearum</i> Isolated from <i>Casuarina</i>.","authors":"Xiaoqing Wang, Chuhao Li, Shaohua Huang, Huagui Gao, Yonglin Li, Xuemei Chen, Liangzhou Huang, Jianhua Luo, LianHui Zhang, Xiaofan Zhou","doi":"10.1094/PDIS-01-24-0118-RE","DOIUrl":"10.1094/PDIS-01-24-0118-RE","url":null,"abstract":"<p><p><i>Casuarina</i> <i>equisetifolia</i> is crucial in protecting coastal regions of China against typhoon attacks but has faced a substantial challenge due to wilt disease caused by pathogens of the <i>Ralstonia solanacearum</i> species complex (RSSC). Although the initial outbreak of <i>Casuarina</i> wilt in the 1970s was effectively controlled by disease-resistant <i>C. equisetifolia</i> varieties, the disease has recently re-emerged in coastal regions of Guangdong. In this study, we report the isolation, characterization, and comparative genomic analysis of 11 RSSC strains from diseased <i>C. equisetifolia</i> at various locations along the coast of Guangdong. Phylogenomic analysis showed that the strains were closely related and clustered with phylotype I strains previously isolated from peanuts. Single-gene-based analysis further suggested these strains could be derived from strains present in Guangdong since the 1980s, indicating a historical context to their current pathogenicity. <i>Casuarina</i>-isolated strains exhibited notably higher virulence against <i>C. equisetifolia</i> and peanuts than the representative RSSC strains GMI1000 and EP1, suggesting host-specific adaptations that possibly contributed to the recent outbreak. Comparative genomic analysis among RSSC strains revealed a largely conserved genome structure and high levels of conservation in gene clusters encoding extracellular polysaccharide biosynthesis, secretion systems, and quorum sensing regulatory systems. However, we also found a number of unique genes in the <i>Casuarina</i>-isolated strains that were absent in GMI1000 and EP1, and vice versa, pointing to potential genetic factors underpinning their differential virulence. These unique genes offer promising targets for future functional studies. Overall, our findings provide crucial insights into the RSSC pathogens causing <i>Casuarina</i> wilt in Guangdong, guiding future efforts in disease control and prevention.</p>","PeriodicalId":20063,"journal":{"name":"Plant disease","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140868934","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-09DOI: 10.1094/PDIS-04-24-0894-RE
David H Gent, Nanci L Adair, Ross J Hatlen, Timothy D Miles, Briana J Richardson, Hannah M Rivedal, Cameron Ross, Michele S Wiseman
Detection and quantification of pathogen propagules in the air or other environmental samples is facilitated by culture-independent assays. We developed a quantitative PCR assay for the hop powdery mildew fungus, Podosphaera macularis, for detection of the organism from air samples. The assay uses primers and a TaqMan probe designed to target species-specific sequences in the 28S large subunit of the nuclear ribosomal DNA. Analytical sensitivity was not affected by the presence of an exogenous internal control or potential PCR inhibitors associated with DNA extracted from soil. The level of quantification of the assay was between 200 and 350 conidia when DNA was extracted from a fixed number of conidia. The assay amplified all isolates of P. macularis tested and had minimal cross-reactivity with other Podosphaera species when assayed with biologically relevant quantities of DNA. Standard curves generated independently in two other laboratories indicated that assay sensitivity was qualitatively similar and reproducible. All laboratories successfully detected eight unknown isolates of P. macularis and correctly discriminated Pseudoperonospora humuli and a water control. The usefulness of the assay for air sampling for late-season inoculum of P. macularis was demonstrated in field studies in 2019 and 2020. In both years, airborne populations of P. macularis in hop yards were detected consistently and increased during bloom and cone development.
与培养无关的检测方法有助于检测和量化空气或其他环境样本中的病原体繁殖体。我们针对啤酒花白粉病真菌 Podosphaera macularis 开发了一种定量 PCR 检测方法，用于检测空气样本中的病原体。该检测方法利用引物和 TaqMan 探针，以核核糖体 rDNA 28S 大亚基（LSU）中的物种特异性序列为目标。分析灵敏度不会受到外源内部对照或与土壤中提取的 DNA 相关的潜在 PCR 抑制剂的影响。从固定数量的分生孢子中提取 DNA 时，检测的定量水平在 200 到 350 个分生孢子之间。当使用生物相关数量的 DNA 进行检测时，该检测方法可扩增所有受测的黄斑疫霉分离株，与其他 Podosphaera 种类的交叉反应极小。另外两个实验室独立生成的标准曲线表明，该检测方法的灵敏度具有相似性和可重复性。所有实验室都成功地检测出了 8 个未知的黄斑癣菌分离物，并正确区分了沼泽假包囊和水对照。2019 年和 2020 年的实地研究证明了该检测方法在空气采样中检测黄斑病菌晚季接种体的实用性。在这两年中，酒花堆场中空气传播的黄斑病菌种群被持续检测到，并且在开花和果实发育期间有所增加。
{"title":"Detection of <i>Podosphaera macularis</i> in Air Samples by Quantitative PCR.","authors":"David H Gent, Nanci L Adair, Ross J Hatlen, Timothy D Miles, Briana J Richardson, Hannah M Rivedal, Cameron Ross, Michele S Wiseman","doi":"10.1094/PDIS-04-24-0894-RE","DOIUrl":"10.1094/PDIS-04-24-0894-RE","url":null,"abstract":"<p><p>Detection and quantification of pathogen propagules in the air or other environmental samples is facilitated by culture-independent assays. We developed a quantitative PCR assay for the hop powdery mildew fungus, <i>Podosphaera macularis</i>, for detection of the organism from air samples. The assay uses primers and a TaqMan probe designed to target species-specific sequences in the 28S large subunit of the nuclear ribosomal DNA. Analytical sensitivity was not affected by the presence of an exogenous internal control or potential PCR inhibitors associated with DNA extracted from soil. The level of quantification of the assay was between 200 and 350 conidia when DNA was extracted from a fixed number of conidia. The assay amplified all isolates of <i>P. macularis</i> tested and had minimal cross-reactivity with other <i>Podosphaera</i> species when assayed with biologically relevant quantities of DNA. Standard curves generated independently in two other laboratories indicated that assay sensitivity was qualitatively similar and reproducible. All laboratories successfully detected eight unknown isolates of <i>P. macularis</i> and correctly discriminated <i>Pseudoperonospora humuli</i> and a water control. The usefulness of the assay for air sampling for late-season inoculum of <i>P. macularis</i> was demonstrated in field studies in 2019 and 2020. In both years, airborne populations of <i>P. macularis</i> in hop yards were detected consistently and increased during bloom and cone development.</p>","PeriodicalId":20063,"journal":{"name":"Plant disease","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140876963","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-05DOI: 10.1094/PDIS-05-24-1048-PDN
Hae Min Lee, Eun Gyeong Song, Ki Hyun Ryu
<p><p>In South Korea, the cultivation area of elephant garlic (Allium ampeloprasum) is increasing as elephant garlic is milder and sweeter than garlic (A. sativum) (Kim et al., 2019; Lu et al., 2011). Viral diseases can decrease garlic productivity by up to 50% in South Korea (Nam et al., 2002). In 2022-2023, virus-like symptoms such as mosaic and yellow stripes were observed on leaves of elephant garlic in a 432㎡farm with disease incidence of approximately 40% in Yangpyeong-gun, Gyeonggi-do, South Korea. Seventy-two leaf samples were randomly collected from symptomatic plants in 2022 (n=46) and 2023 (n=26). Total RNAs were isolated from individual samples using the Total RNA Prep Kit (BioFact, Daejeon, Korea), and then two-steps RT-PCR was performed using the First Strand cDNA Synthesis kit (Thermo Fisher Scientific) and the TaKaRa TaqTM (TaKaRa Bio Inc.). These samples were tested for 13 viruses with virus-specific coat protein primers including garlic common latent virus (GarCLV) (supplementary Table 1). In 2022, GarCLV, garlic virus (GarV)-B, GarV-C, and GarV-D were detected with the expected amplicon sizes of their CP genes (960, 735, 780, and 753 bp, respectively) in four different plants. In 2023, the CP gene of GarCLV was detected in 26 samples and 4 of 26 samples were positive for GarV-B. The leaves infected with GarCLV and GarV-B in mixed infection showed synergistic effect with extended mosaic and yellow stripes than the leaves with single infection (supplementary Fig. 1). All amplicons were cloned into a pGEM-T Easy vector (Promega Co., USA), and sequenced at Bionics Co. Ltd., South Korea. The resulting nucleotide (nt) and amino acid (aa) sequences were analyzed using DNAMAN software version 5.1. Since all isolates were collected from a farm in Yangpyeong-gun, name of these isolates started with "YPG." The nt and aa sequences of the isolates were compared with those of other strains/isolates. All 27 GarCLV-YPG isolates sequences were deposited (Accessions: OP981636, and PP533185-PP533210). The GarCLV-YPG sequences shared 78.90%-94.40% nt and 92.10%-99.40% aa identities with other GarCLV strains and isolates, and they showed higher similarity (99.40% aa) to isolates produced from A. sativum in China and India (supplementary Table 2). GarV-C-YPG showed the highest similarity (99.20% aa) to isolate G81(GenBank MN059141) from A. sativum in China. GarV-D-YPG showed the highest similarity (99.20% aa) to isolates (G82, GenBank MN059388; BR, MT279193) from A. sativum in China and Brazil. Twenty-two quinoa plants (Chenopodium quinoa, local lesion host) were individually inoculated using the sap from 22 GarCLV infected plants. Chlorotic and necrotic spots appeared on inoculated leaves 12 days post-inoculation; no chlorotic and necrotic spots symptoms were observed on any other leaves except for the inoculated leaves. RT-PCR was performed and the targeted amplicon size for GarCLV was detected. In transmission electron microscope, filamentous par
{"title":"First report of garlic common latent virus in elephant garlic (<i>Allium ampeloprasum</i>) in single and mixed infection in South Korea.","authors":"Hae Min Lee, Eun Gyeong Song, Ki Hyun Ryu","doi":"10.1094/PDIS-05-24-1048-PDN","DOIUrl":"https://doi.org/10.1094/PDIS-05-24-1048-PDN","url":null,"abstract":"<p><p>In South Korea, the cultivation area of elephant garlic (Allium ampeloprasum) is increasing as elephant garlic is milder and sweeter than garlic (A. sativum) (Kim et al., 2019; Lu et al., 2011). Viral diseases can decrease garlic productivity by up to 50% in South Korea (Nam et al., 2002). In 2022-2023, virus-like symptoms such as mosaic and yellow stripes were observed on leaves of elephant garlic in a 432㎡farm with disease incidence of approximately 40% in Yangpyeong-gun, Gyeonggi-do, South Korea. Seventy-two leaf samples were randomly collected from symptomatic plants in 2022 (n=46) and 2023 (n=26). Total RNAs were isolated from individual samples using the Total RNA Prep Kit (BioFact, Daejeon, Korea), and then two-steps RT-PCR was performed using the First Strand cDNA Synthesis kit (Thermo Fisher Scientific) and the TaKaRa TaqTM (TaKaRa Bio Inc.). These samples were tested for 13 viruses with virus-specific coat protein primers including garlic common latent virus (GarCLV) (supplementary Table 1). In 2022, GarCLV, garlic virus (GarV)-B, GarV-C, and GarV-D were detected with the expected amplicon sizes of their CP genes (960, 735, 780, and 753 bp, respectively) in four different plants. In 2023, the CP gene of GarCLV was detected in 26 samples and 4 of 26 samples were positive for GarV-B. The leaves infected with GarCLV and GarV-B in mixed infection showed synergistic effect with extended mosaic and yellow stripes than the leaves with single infection (supplementary Fig. 1). All amplicons were cloned into a pGEM-T Easy vector (Promega Co., USA), and sequenced at Bionics Co. Ltd., South Korea. The resulting nucleotide (nt) and amino acid (aa) sequences were analyzed using DNAMAN software version 5.1. Since all isolates were collected from a farm in Yangpyeong-gun, name of these isolates started with \"YPG.\" The nt and aa sequences of the isolates were compared with those of other strains/isolates. All 27 GarCLV-YPG isolates sequences were deposited (Accessions: OP981636, and PP533185-PP533210). The GarCLV-YPG sequences shared 78.90%-94.40% nt and 92.10%-99.40% aa identities with other GarCLV strains and isolates, and they showed higher similarity (99.40% aa) to isolates produced from A. sativum in China and India (supplementary Table 2). GarV-C-YPG showed the highest similarity (99.20% aa) to isolate G81(GenBank MN059141) from A. sativum in China. GarV-D-YPG showed the highest similarity (99.20% aa) to isolates (G82, GenBank MN059388; BR, MT279193) from A. sativum in China and Brazil. Twenty-two quinoa plants (Chenopodium quinoa, local lesion host) were individually inoculated using the sap from 22 GarCLV infected plants. Chlorotic and necrotic spots appeared on inoculated leaves 12 days post-inoculation; no chlorotic and necrotic spots symptoms were observed on any other leaves except for the inoculated leaves. RT-PCR was performed and the targeted amplicon size for GarCLV was detected. In transmission electron microscope, filamentous par","PeriodicalId":20063,"journal":{"name":"Plant disease","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142133437","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-05DOI: 10.1094/PDIS-06-24-1227-PDN
Honglong Chu, Kun Dong, Yong Gao, Zhao Liu, Jie Huang, Yanan Zhang
<p><p>Amorphophallus konjac, commonly called voodoo lily, is a cash crop widely cultivated in southwest China (Gao et al. 2022). In August 2022, leaf spot symptoms were observed in a field (1 ha) located at Fuyuan (25.67°N; 104.25°E), Yunnan, China, resulting in substantial economic losses. Brown lesions, with an incidence ranging from 20 to 40%, typically had a whitish or gray center and were surrounded by yellow halos. Microscopic observations of the spots revealed anamorphic species Cercospora chevalieri. Conidiophores were 50-150 × 4-7 μm, cylindrical, unbranched, smooth-walled, pale brown and aggregated in dense fascicles arising from a brown stroma. The conidiogenous cells were integrated, terminal or intercalary, pale brown to brown and proliferated sympodially. The conidiogenous loci were thickened and darkened, and 2-3 μm in diam. The conidia were formed singly, obclavate-cylindrical, 90-160 × 5-7 μm, with an average of 130 × 6 μm (n = 30), 6-11 septa, thin-walled, smooth, hyaline or subhyaline, straight or curved with an obtuse apex and obconically truncate base, with thickened and darkened hilum. These morphological characteristics matched those of C. chevalieri, the causal agent of leaf spot on A. paeoniifolius (Braun et al. 2014; Saccardo et al. 1913). A conidial suspension in sterile water from lesions was used to inoculate water agar, and germinated conidia were transferred to potato dextrose agar（PDA) and incubated at 27°C for 7 days. Induction of sporulation was unsuccessful using PDA, as well as malt extract agar, potato sucrose agar and synthetic nutrient-poor agar. Two out of ten isolates were selected for molecular identification and pathogenicity assay. Genomic DNA from two pure isolates (KUNCC22-12536 and KUNCC22-12537) was extracted for PCR and amplified with primers for the internal transcribed spacers (ITS: ITS1/ITS4), calmodulin (CMD: CAL228F/CAL2Rd), translation elongation factor 1-alpha (TEF1-α: 728F/986R), actin (ACT: 512F/783R), histone H3 (HIS3: CYLH3F/CYLH3R), beta-tubulin gene (TUB2: BT-1F/BT-1R) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH: Gpd1/Gpd2), respectively (Vaghefi et al. 2021). The newly generated sequences for ITS (OP719153/OP719154), CMD(OP740904/OP740905), TEF1-α (OP740910/OP740911), ACT (OP740902/OP740903), HIS3 (OP740908/OP740909), TUB2 (OP740912/OP740913), GAPDH (OP740906/OP740907) of C. chevalieri were submitted to GenBank. So far, no sequence data of C. chevalieri were available in the GenBank database. As expected, most genes (TEF1-α, ACT, CMD, HIS, TUB2 and GAPDH) showed 91 to 95% identity to their best hits within species of the genus Cercospora. The phylogenetic tree showed that sequences retrieved from two isolates obtained from the A. konjac leaf spots clustered together within Cercospora forming a strongly supported clade. To test Koch's postulates, ten four-month-old healthy A. konjac plants grown in pots were used for a pathogenicity test in a greenhouse. One leaf of each plant
{"title":"First Report of <i>Cercospora chevalieri</i> Causing A Leaf Spot Disease on <i>Amorphophallus konjac</i> in China.","authors":"Honglong Chu, Kun Dong, Yong Gao, Zhao Liu, Jie Huang, Yanan Zhang","doi":"10.1094/PDIS-06-24-1227-PDN","DOIUrl":"https://doi.org/10.1094/PDIS-06-24-1227-PDN","url":null,"abstract":"<p><p>Amorphophallus konjac, commonly called voodoo lily, is a cash crop widely cultivated in southwest China (Gao et al. 2022). In August 2022, leaf spot symptoms were observed in a field (1 ha) located at Fuyuan (25.67°N; 104.25°E), Yunnan, China, resulting in substantial economic losses. Brown lesions, with an incidence ranging from 20 to 40%, typically had a whitish or gray center and were surrounded by yellow halos. Microscopic observations of the spots revealed anamorphic species Cercospora chevalieri. Conidiophores were 50-150 × 4-7 μm, cylindrical, unbranched, smooth-walled, pale brown and aggregated in dense fascicles arising from a brown stroma. The conidiogenous cells were integrated, terminal or intercalary, pale brown to brown and proliferated sympodially. The conidiogenous loci were thickened and darkened, and 2-3 μm in diam. The conidia were formed singly, obclavate-cylindrical, 90-160 × 5-7 μm, with an average of 130 × 6 μm (n = 30), 6-11 septa, thin-walled, smooth, hyaline or subhyaline, straight or curved with an obtuse apex and obconically truncate base, with thickened and darkened hilum. These morphological characteristics matched those of C. chevalieri, the causal agent of leaf spot on A. paeoniifolius (Braun et al. 2014; Saccardo et al. 1913). A conidial suspension in sterile water from lesions was used to inoculate water agar, and germinated conidia were transferred to potato dextrose agar（PDA) and incubated at 27°C for 7 days. Induction of sporulation was unsuccessful using PDA, as well as malt extract agar, potato sucrose agar and synthetic nutrient-poor agar. Two out of ten isolates were selected for molecular identification and pathogenicity assay. Genomic DNA from two pure isolates (KUNCC22-12536 and KUNCC22-12537) was extracted for PCR and amplified with primers for the internal transcribed spacers (ITS: ITS1/ITS4), calmodulin (CMD: CAL228F/CAL2Rd), translation elongation factor 1-alpha (TEF1-α: 728F/986R), actin (ACT: 512F/783R), histone H3 (HIS3: CYLH3F/CYLH3R), beta-tubulin gene (TUB2: BT-1F/BT-1R) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH: Gpd1/Gpd2), respectively (Vaghefi et al. 2021). The newly generated sequences for ITS (OP719153/OP719154), CMD(OP740904/OP740905), TEF1-α (OP740910/OP740911), ACT (OP740902/OP740903), HIS3 (OP740908/OP740909), TUB2 (OP740912/OP740913), GAPDH (OP740906/OP740907) of C. chevalieri were submitted to GenBank. So far, no sequence data of C. chevalieri were available in the GenBank database. As expected, most genes (TEF1-α, ACT, CMD, HIS, TUB2 and GAPDH) showed 91 to 95% identity to their best hits within species of the genus Cercospora. The phylogenetic tree showed that sequences retrieved from two isolates obtained from the A. konjac leaf spots clustered together within Cercospora forming a strongly supported clade. To test Koch's postulates, ten four-month-old healthy A. konjac plants grown in pots were used for a pathogenicity test in a greenhouse. One leaf of each plant ","PeriodicalId":20063,"journal":{"name":"Plant disease","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142140785","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-05DOI: 10.1094/PDIS-06-24-1182-PDN
Mengjia Xu, Hao Ai, Rong Yan, Pengbo Li, Ruixiang Cheng, Yi Xu
<p><p>Sesame (<i>Sesamum indicum</i> L.) is one of the primary oilseed crops in China, and often intercropped with shorter crops like peanuts and soybeans. Cowpea mild mottle virus (CpMMV), a member of the <i>Betaflexiviridae</i> family, has been reported in numerous countries worldwide and can infect natural hosts including cowpeas, soybeans, common beans, peanuts, and tomatoes, causing symptoms such as leaf mottling, mosaic patterns, or spotted patterns on the infected leaves. CpMMV is transmitted by whiteflies in nature and by mechanical inoculation in laboratory settings (Iwaki et al., 1982). In September 2023, while surveying soybean virus diseases in Huang-Huai-Hai region of China, we observed sesame plants near a soybean field (longitude 115.76°E, latitude 32.89°N) showing stunted growth, leaf mottling, and mosaic patterns. These symptoms affected approximately one-third of the sesame plants in a 0.1-hectare field. To identify the virus associated with symptomatic leaves, two sesame samples were collected for small RNA deep sequencing. Total RNA was extracted using TRIZOL and sent to BGI for library construction and sequencing with the BGISEQ-500 sequencer. De novo assembly of sRNA reads was performed using Velvet software (version 1.2.10) as described (Su et al., 2016), followed by BLASTn and BLASTx searches against the nonredundant nucleotide and protein databases. CpMMV was identified from sesame plants, with twenty-three contigs ranging from 51 to 368 nucleotides showing similarity to CpMMV, covering 33.7% of the total CpMMV genome. The largest CpMMV contig, spanning 368 nucleotides (nt), exhibited 97% identity to CpMMV isolate Anhui_SZ_DN1383 (Genbank Accession No. MN908944.1) from soybean (Wei et al., 2020). To validate the presence of CpMMV in sesame, RNA from each sample was individually extracted, and CpMMV was detected by reverse-transcription polymerase chain reaction (RT-PCR) according to the manufacturer's instructions (Vazyme, Nanjing, China). Primers were designed based on two small RNA-assembled contigs spanning the CpMMV triple gene block protein 1 (TGBp1) and TGBp2 ORF (Forward: 5´-GGTACCAAAAGATAAGCTTGTTATCTTG-3´; Reverse: 5´-TTAGTACCGTCTCTGTAACAGCCA-3´). Both sesame samples tested RT-PCR positive for CpMMV. The PCR amplicon (597 nt) of these two sesame samples were purified and sequenced. Sequences shared 100% nucleotide identity between them. Nucleotide sequence comparisons confirmed the virus as CpMMV (Accession No. PP767740), exhibiting >99% identity to CpMMV isolate HN_SQ (MW354940.1). Phylogenetic analysis of the 597 nt amplicon, using MEGA7 with eighteen other CpMMV isolates, revealed that the CpMMV isolate from sesame was most closely related to soybean isolates HN_SQ (MW354940.1) and Anhui_SZ_DN1383 (MN908944.1). To fulfill Koch's postulates, healthy sesame leaves were rub-inoculated with crude extracts from CpMMV-infected field samples. RT-PCR confirmed systemic infection at 4 weeks post-inoculation, with sympto
{"title":"Natural Occurrence of cowpea mild mottle virus infecting sesame (<i>Sesamum indicum</i> L.) in Anhui Province, China.","authors":"Mengjia Xu, Hao Ai, Rong Yan, Pengbo Li, Ruixiang Cheng, Yi Xu","doi":"10.1094/PDIS-06-24-1182-PDN","DOIUrl":"https://doi.org/10.1094/PDIS-06-24-1182-PDN","url":null,"abstract":"<p><p>Sesame (<i>Sesamum indicum</i> L.) is one of the primary oilseed crops in China, and often intercropped with shorter crops like peanuts and soybeans. Cowpea mild mottle virus (CpMMV), a member of the <i>Betaflexiviridae</i> family, has been reported in numerous countries worldwide and can infect natural hosts including cowpeas, soybeans, common beans, peanuts, and tomatoes, causing symptoms such as leaf mottling, mosaic patterns, or spotted patterns on the infected leaves. CpMMV is transmitted by whiteflies in nature and by mechanical inoculation in laboratory settings (Iwaki et al., 1982). In September 2023, while surveying soybean virus diseases in Huang-Huai-Hai region of China, we observed sesame plants near a soybean field (longitude 115.76°E, latitude 32.89°N) showing stunted growth, leaf mottling, and mosaic patterns. These symptoms affected approximately one-third of the sesame plants in a 0.1-hectare field. To identify the virus associated with symptomatic leaves, two sesame samples were collected for small RNA deep sequencing. Total RNA was extracted using TRIZOL and sent to BGI for library construction and sequencing with the BGISEQ-500 sequencer. De novo assembly of sRNA reads was performed using Velvet software (version 1.2.10) as described (Su et al., 2016), followed by BLASTn and BLASTx searches against the nonredundant nucleotide and protein databases. CpMMV was identified from sesame plants, with twenty-three contigs ranging from 51 to 368 nucleotides showing similarity to CpMMV, covering 33.7% of the total CpMMV genome. The largest CpMMV contig, spanning 368 nucleotides (nt), exhibited 97% identity to CpMMV isolate Anhui_SZ_DN1383 (Genbank Accession No. MN908944.1) from soybean (Wei et al., 2020). To validate the presence of CpMMV in sesame, RNA from each sample was individually extracted, and CpMMV was detected by reverse-transcription polymerase chain reaction (RT-PCR) according to the manufacturer's instructions (Vazyme, Nanjing, China). Primers were designed based on two small RNA-assembled contigs spanning the CpMMV triple gene block protein 1 (TGBp1) and TGBp2 ORF (Forward: 5´-GGTACCAAAAGATAAGCTTGTTATCTTG-3´; Reverse: 5´-TTAGTACCGTCTCTGTAACAGCCA-3´). Both sesame samples tested RT-PCR positive for CpMMV. The PCR amplicon (597 nt) of these two sesame samples were purified and sequenced. Sequences shared 100% nucleotide identity between them. Nucleotide sequence comparisons confirmed the virus as CpMMV (Accession No. PP767740), exhibiting >99% identity to CpMMV isolate HN_SQ (MW354940.1). Phylogenetic analysis of the 597 nt amplicon, using MEGA7 with eighteen other CpMMV isolates, revealed that the CpMMV isolate from sesame was most closely related to soybean isolates HN_SQ (MW354940.1) and Anhui_SZ_DN1383 (MN908944.1). To fulfill Koch's postulates, healthy sesame leaves were rub-inoculated with crude extracts from CpMMV-infected field samples. RT-PCR confirmed systemic infection at 4 weeks post-inoculation, with sympto","PeriodicalId":20063,"journal":{"name":"Plant disease","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142133440","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-05DOI: 10.1094/PDIS-06-24-1161-RE
Pei-Hsin Lo, Jin-Hsing Huang, Chia-Chih Chang, Ahmed Namisy, Chi Yu Chen, Wen-Hsin Chung
Fusarium solani species complex (FSSC) is a causal agent of collar rot and fruit rot in passion fruit worldwide. This study investigated the diversity and characteristics of FSSC isolates causing collar rot and fruit rot in Taiwanese passion fruit. Thirty-five FSSC isolates were harvested from collar rot and fruit rot samples of passion fruit from various cultivars and different geographical locations in Taiwan. The majority of these FSSC isolates caused collar rot and fruit rot disease of varying virulence in the stems and fruits of the purple and yellow cultivars of passion fruit. FSSC isolates were categorized into four groups: F. solani-melongenae (FSSC 21; n=29), F. solani (FSSC 5; n=1), F. liriodendri (FSSC 24; n=1), and an unknown group (n=4) based on the phylogenetic analysis of internal transcribed sequence (ITS), translation elongation factor 1 alpha (TEF-1α), and RNA polymerase II subunit 2 (RPB2) sequences. In Taiwan, F. solani-melongenae was the dominant species causing collar rot and fruit rot in passion fruit. F. solani-melongenae was a homothallic fungus that produced perithecia in diseased tissues. However, F. solani and F. liriodendri did not produce perithecia. The unknown FSSC group showed morphological characteristics similar to F. solani-melongenae and produced perithecia. Phylogenetic analysis based on the ITS and TEF-1α sequences demonstrated that the Taiwanese FSSC isolates were distinct from the Brazilian and Chinese FSSC isolates. In summary, FSSC isolates causing collar rot and fruit rot of Taiwanese passion fruit showed high diversity, potentially associated with the geographical locations.
Fusarium solani species complex（FSSC）是全球百香果领腐病和果腐病的病原菌。本研究调查了引起台湾百香果领腐病和果腐病的 FSSC 分离物的多样性和特征。研究人员从台湾不同栽培品种和不同地理位置的百香果领腐病和果腐病样本中提取了 35 株 FSSC 分离物。这些 FSSC 分离物大多对紫色和黄色百香果栽培品种的茎和果实造成不同程度的领腐病和果腐病。FSSC 分离物被分为四类：F.solani-melongenae（FSSC 21；n=29）、F.solani（FSSC 5；n=1）、F.liriodendri（FSSC 24；n=1）和一个未知组（n=4），其依据是内部转录序列（ITS）、翻译延伸因子 1 alpha（TEF-1α）和 RNA 聚合酶 II 亚基 2（RPB2）序列的系统发育分析。在台湾，F. solani-melongenae是导致百香果领腐病和果腐病的主要物种。F.solani-melongenae是一种同室真菌，在病组织中产生包囊。然而，F. solani 和 F. liriodendri 不产生珠孔。未知的 FSSC 群体表现出与 F. solani-melongenae 相似的形态特征，并能产生珠菌。基于 ITS 和 TEF-1α 序列的系统发育分析表明，台湾的 FSSC 分离物与巴西和中国的 FSSC 分离物不同。总之，导致台湾百香果领腐病和果腐病的 FSSC 分离物表现出高度的多样性，这可能与地理位置有关。
{"title":"Diversity and characteristics of <i>Fusarium solani</i> species complex (FSSC) isolates causing collar rot and fruit rot of passion fruit in Taiwan.","authors":"Pei-Hsin Lo, Jin-Hsing Huang, Chia-Chih Chang, Ahmed Namisy, Chi Yu Chen, Wen-Hsin Chung","doi":"10.1094/PDIS-06-24-1161-RE","DOIUrl":"https://doi.org/10.1094/PDIS-06-24-1161-RE","url":null,"abstract":"<p><p><i>Fusarium solani</i> species complex (FSSC) is a causal agent of collar rot and fruit rot in passion fruit worldwide. This study investigated the diversity and characteristics of FSSC isolates causing collar rot and fruit rot in Taiwanese passion fruit. Thirty-five FSSC isolates were harvested from collar rot and fruit rot samples of passion fruit from various cultivars and different geographical locations in Taiwan. The majority of these FSSC isolates caused collar rot and fruit rot disease of varying virulence in the stems and fruits of the purple and yellow cultivars of passion fruit. FSSC isolates were categorized into four groups: <i>F. solani-melongenae</i> (FSSC 21; n=29), <i>F. solani</i> (FSSC 5; n=1), <i>F. liriodendri</i> (FSSC 24; n=1), and an unknown group (n=4) based on the phylogenetic analysis of internal transcribed sequence (ITS), translation elongation factor 1 alpha (TEF-1<i>α</i>), and RNA polymerase II subunit 2 (RPB2) sequences. In Taiwan, <i>F. solani-melongenae</i> was the dominant species causing collar rot and fruit rot in passion fruit. <i>F. solani-melongenae</i> was a homothallic fungus that produced perithecia in diseased tissues. However, <i>F. solani</i> and <i>F. liriodendri</i> did not produce perithecia. The unknown FSSC group showed morphological characteristics similar to <i>F. solani-melongenae</i> and produced perithecia. Phylogenetic analysis based on the ITS and TEF-1<i>α</i> sequences demonstrated that the Taiwanese FSSC isolates were distinct from the Brazilian and Chinese FSSC isolates. In summary, FSSC isolates causing collar rot and fruit rot of Taiwanese passion fruit showed high diversity, potentially associated with the geographical locations.</p>","PeriodicalId":20063,"journal":{"name":"Plant disease","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142133435","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
This study aimed to investigate the Diaporthe species associated with Phomopsis stem canker of sunflower (Helianthus annuus L.) in Serbia. The significant increase in sunflower and soybean (Glycine max [L.] Merr.) cultivation may have created the bridge favorable conditions for the distribution of Diaporthe species in this region. The present study identified five Diaporthe species on sunflower: D. gulyae, D. helianthi, D. pseudolongicolla, D. stewartii, and the newly identified D. riccionae based on morphological, molecular, and pathogenic characteristics. The research emphasizes the importance of effective inoculation methods and evaluates the aggressiveness of isolates. Sunflower plants were inoculated using the stem wound method, while seeds of sunflower and soybean were inoculated using the standard seed method. Most of the tested isolates demonstrated high aggressiveness, resulting in more than 80% premature wilting of sunflower plants. Additionally, this research examined the aggressiveness of Diaporthe species on sunflower seeds, highlighting D. stewartii and D. pseudolongicolla as common pathogens of both sunflower and soybean. The most aggressive species on seeds was D. stewartii, causing seed decay of up to 100% in sunflower and 97% in soybean. The findings suggest the development of resilient sunflower genotypes through breeding programs and the implementation of strategies to manage cross-contamination risks between sunflower and soybean crops. Furthermore, this study provides insights into the interactions between Diaporthe species and the seeds of sunflower and soybean. Future research will enhance our understanding of the impact of Diaporthe species on sunflower and soybean.
本研究旨在调查与塞尔维亚向日葵（Helianthus annuus L.）茎腐病拟南芥相关的 Diaporthe 物种。向日葵和大豆（Glycine max (L.) Merr.）种植的大幅增加可能为 Diaporthe 物种在该地区的分布创造了有利条件。本研究确定了向日葵上的五个 Diaporthe 物种：D. gulyae、D. helianthi、D. pseudolongicolla、D. stewartii 和新发现的 D. riccionae。研究强调了有效接种方法的重要性，并评估了分离株的侵染性。向日葵植株采用茎伤法接种，向日葵和大豆种子则采用标准种子法接种。大多数测试的分离物都表现出很强的侵染性，导致向日葵植株 80% 以上过早枯萎。此外，这项研究还考察了向日葵种子上的 Diaporthe 物种的侵染性，发现 D. stewartii 和 D. pseudolongicolla 是向日葵和大豆的常见病原体。对种子最具侵袭性的病原菌是 D. stewartii，它对向日葵种子的腐烂率高达 100%，对大豆的腐烂率高达 97%。研究结果表明，应通过育种计划开发抗逆性强的向日葵基因型，并实施策略管理向日葵和大豆作物之间的交叉污染风险。此外，这项研究还为 Diaporthe 物种与向日葵和大豆种子之间的相互作用提供了见解。未来的研究将加深我们对 Diaporthe 物种对向日葵和大豆影响的了解。
{"title":"Diversity and Aggressiveness of the <i>Diaporthe</i> Species Complex on Sunflower in Serbia.","authors":"Slobodan Krsmanović, Luca Riccioni, Boško Dedić, Febina Merlin Mathew, Miodrag Tolimir, Vera Stojšin, Kristina Petrović","doi":"10.1094/PDIS-01-24-0195-RE","DOIUrl":"10.1094/PDIS-01-24-0195-RE","url":null,"abstract":"<p><p>This study aimed to investigate the <i>Diaporthe</i> species associated with Phomopsis stem canker of sunflower (<i>Helianthus annuus</i> L.) in Serbia. The significant increase in sunflower and soybean (<i>Glycine max</i> [L.] Merr.) cultivation may have created the bridge favorable conditions for the distribution of <i>Diaporthe</i> species in this region. The present study identified five <i>Diaporthe</i> species on sunflower: <i>D. gulyae</i>, <i>D. helianthi</i>, <i>D. pseudolongicolla</i>, <i>D. stewartii</i>, and the newly identified <i>D. riccionae</i> based on morphological, molecular, and pathogenic characteristics. The research emphasizes the importance of effective inoculation methods and evaluates the aggressiveness of isolates. Sunflower plants were inoculated using the stem wound method, while seeds of sunflower and soybean were inoculated using the standard seed method. Most of the tested isolates demonstrated high aggressiveness, resulting in more than 80% premature wilting of sunflower plants. Additionally, this research examined the aggressiveness of <i>Diaporthe</i> species on sunflower seeds, highlighting <i>D. stewartii</i> and <i>D. pseudolongicolla</i> as common pathogens of both sunflower and soybean. The most aggressive species on seeds was <i>D. stewartii</i>, causing seed decay of up to 100% in sunflower and 97% in soybean. The findings suggest the development of resilient sunflower genotypes through breeding programs and the implementation of strategies to manage cross-contamination risks between sunflower and soybean crops. Furthermore, this study provides insights into the interactions between <i>Diaporthe</i> species and the seeds of sunflower and soybean. Future research will enhance our understanding of the impact of <i>Diaporthe</i> species on sunflower and soybean.</p>","PeriodicalId":20063,"journal":{"name":"Plant disease","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140870858","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-05DOI: 10.1094/PDIS-07-24-1416-PDN
Okhee Choi, Haeun Noh, Yeyeong Lee, Jinwoo Kim
<p><p>Winter squash (Cucurbita maxima) is rich in vitamins C and B6 and is also a source of beta-carotene, a provitamin A carotenoid. About 13,000 tons have been produced annually in South Korea over the past 10 years. In the summer of 2022, severe rot was observed in winter squash for sale at a wholesale market in Jinju, South Korea, with approximately 10% of the 500 squashes observed affected. White fungal hyphae and dark orange spore masses were observed on the surface of the decayed squash. To isolate the causal agents, symptomatic tissues (3 × 3 mm) between diseased and healthy tissues per squash from 3 diseased squashes were excised, disinfested with 1% sodium hypochlorite for 20 s and 70% ethanol for 10 s, washed twice in sterilized distilled water, dried on sterilized filter paper, transferred to water agar, and incubated at 25°C for 2 days. Agar blocks (3 mm<sup>2</sup>) containing fungal colonies were transferred to potato dextrose agar (PDA) plates and incubated at 25°C until fungal colonies grew. Three isolates (GNU F137a‒c) with similar morphology were subcultured using the single-spore method. In PDA, the colonies looked like gray cotton when viewed from the front, were pale orange from the back, and numerous small black sclerotia-like grains could be observed on both sides. Setae were pale to medium brown, verrucose, 40-120 μm long, and 3-6 septated. Conidiophores were hyaline to pale brown, smooth-walled, septate, branched, and up to 45 μm long. Conidia were hyaline, smooth walled, aseptate, straight, cylindrical, the apex and base rounded, and 14-18 × 5-7 μm (n = 30). Appressoria were single, brown, aseptate, ellipsoidal to irregular in outline, with crenate margins, and 3.5-5 × 3-5 μm (n = 30). The morphological features of the fungal isolates matched descriptions of Colletotrichum species. To confirm the identity of the isolated fungus, genomic DNA of all three isolates was extracted using the Phire Plant Direct PCR Kit (Thermo Fisher Scientific, Baltics, UAB). The internal transcribed spacers (ITS) of the ribosomal RNA gene region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), histone H3 (HIS3), actin (ACT), and beta-tubulin (TUB2) genes were amplified and sequenced using the primer pairs ITS1/ITS4, GDF/GDR, CHS-79F/CHS-354R, CYLH3F/CYLH3R, ACT-512F/ACT-783R, and T1/T2, respectively. The sequences were deposited in GenBank (acc. nos., PP504320 and PP555649-PP555653). Concatenated sequences of the six genes obtained from isolates GNU F137a‒c and ex-types from each accepted taxon in previous studies were used to conduct a phylogenetic analysis using the maximum likelihood method in MEGA 11. The fungus isolated from winter squash was in the same clade as C. liaoningense. Therefore, the isolates were identified as C. liaoningense. For pathogenicity tests, three winter squash were wounded with a sterilized needle and inoculated with each isolate by injecting 100 μl conidial suspension (10<sup>5</sup>
{"title":"First report of <i>Colletotrichum liaoningense</i> causing anthracnose on winter squash in Korea.","authors":"Okhee Choi, Haeun Noh, Yeyeong Lee, Jinwoo Kim","doi":"10.1094/PDIS-07-24-1416-PDN","DOIUrl":"https://doi.org/10.1094/PDIS-07-24-1416-PDN","url":null,"abstract":"<p><p>Winter squash (Cucurbita maxima) is rich in vitamins C and B6 and is also a source of beta-carotene, a provitamin A carotenoid. About 13,000 tons have been produced annually in South Korea over the past 10 years. In the summer of 2022, severe rot was observed in winter squash for sale at a wholesale market in Jinju, South Korea, with approximately 10% of the 500 squashes observed affected. White fungal hyphae and dark orange spore masses were observed on the surface of the decayed squash. To isolate the causal agents, symptomatic tissues (3 × 3 mm) between diseased and healthy tissues per squash from 3 diseased squashes were excised, disinfested with 1% sodium hypochlorite for 20 s and 70% ethanol for 10 s, washed twice in sterilized distilled water, dried on sterilized filter paper, transferred to water agar, and incubated at 25°C for 2 days. Agar blocks (3 mm<sup>2</sup>) containing fungal colonies were transferred to potato dextrose agar (PDA) plates and incubated at 25°C until fungal colonies grew. Three isolates (GNU F137a‒c) with similar morphology were subcultured using the single-spore method. In PDA, the colonies looked like gray cotton when viewed from the front, were pale orange from the back, and numerous small black sclerotia-like grains could be observed on both sides. Setae were pale to medium brown, verrucose, 40-120 μm long, and 3-6 septated. Conidiophores were hyaline to pale brown, smooth-walled, septate, branched, and up to 45 μm long. Conidia were hyaline, smooth walled, aseptate, straight, cylindrical, the apex and base rounded, and 14-18 × 5-7 μm (n = 30). Appressoria were single, brown, aseptate, ellipsoidal to irregular in outline, with crenate margins, and 3.5-5 × 3-5 μm (n = 30). The morphological features of the fungal isolates matched descriptions of Colletotrichum species. To confirm the identity of the isolated fungus, genomic DNA of all three isolates was extracted using the Phire Plant Direct PCR Kit (Thermo Fisher Scientific, Baltics, UAB). The internal transcribed spacers (ITS) of the ribosomal RNA gene region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), histone H3 (HIS3), actin (ACT), and beta-tubulin (TUB2) genes were amplified and sequenced using the primer pairs ITS1/ITS4, GDF/GDR, CHS-79F/CHS-354R, CYLH3F/CYLH3R, ACT-512F/ACT-783R, and T1/T2, respectively. The sequences were deposited in GenBank (acc. nos., PP504320 and PP555649-PP555653). Concatenated sequences of the six genes obtained from isolates GNU F137a‒c and ex-types from each accepted taxon in previous studies were used to conduct a phylogenetic analysis using the maximum likelihood method in MEGA 11. The fungus isolated from winter squash was in the same clade as C. liaoningense. Therefore, the isolates were identified as C. liaoningense. For pathogenicity tests, three winter squash were wounded with a sterilized needle and inoculated with each isolate by injecting 100 μl conidial suspension (10<sup>5</sup> ","PeriodicalId":20063,"journal":{"name":"Plant disease","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142140786","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-05DOI: 10.1094/PDIS-07-24-1512-PDN
Amanda Penicks, Jakob D Johnson, Annette L Wszelaki, Leslie L Domier, M R Hajimorad
<p><p>In the summer of 2021, a field survey of several tomato-growing counties in Tennessee (TN) was conducted for plants exhibiting virus-like symptoms. While scouting in September in Grainger County, one of the largest areas under tomato (Solanum lycopersicum) production in TN, leaves from six tomato plants (cv. BHN 589) growing on a farm located near Rutledge were collected and subsequently stored at -80˚C. Only one of the plants exhibited symptoms typical of tomato yellow leaf curl virus (TYLCV) infection, which included chlorosis, leaf curling, downward cupping, thickening, and mottling. Total DNA was isolated using the DNeasy Plant Mini Kit (Qiagen, Santa Clara, CA) and subjected to PCR using primers TYv2337F (5'-ACGTAGGTCTTGACATCTGTTGAGCTC-3') and TYc138-R: (5'-AAGTGGGTCCCACAATTGCAAGAC-3') and Ex-Taq polymerase (Takara Bio, Mountain View, CA) to amplify a 634-bp genomic fragment of TYLCV (Alkowni et al. 2019). Primers against tomato elongation factor-1 served as internal PCR control (Dias et al. 2023). Each primer set amplified amplicons of expected sizes; however, the TYLCV fragment was detected only from the plant exhibiting typical symptoms of infection. Amplicons were purified with the QIAquick PCR purification kit (Qiagen) and sequenced directly bi-directionally by Eurofins USA using the above primers. The resultant sequences were edited and analyzed with CLC Genomic Workbench v. 24.0.1. Blast analysis of the sequences (606 nts) against those available in GenBank showed 93 TYLCV isolates with over 95% nucleotide sequence identity. Subsequently, the full-length genome was PCR amplified using primers TYBamHIv (5'- GGATCCACTTCTAAATGAATTTCCTG-3') and TYBamHI2c (5'-GGATCCCACATAGTGCAAGACAAAC-3') (Rojas et al. 2007), ligated into pGEM-T (Promega, Madison, WI) and cloned. Plasmids were purified using QIAprep Spin Miniprep kit (Qiagen) and five independent plasmids clones were sequenced using Oxford Nanopore sequencing (v14 library chemistry & R10.4.1 flow cell) by Eurofins USA. The resultant sequences were edited and analyzed with CLC Genomic Workbench and a consensus sequence representing the full-length genome (2,781 nts) was generated and submitted to GenBank (Accession No. PP505780). Blast analysis showed over 98% nucleotide sequence identity with 100 TYLCV isolates from GenBank. The highest sequence identity of 98.6% was with the sequence of an isolate from Florida (AY530931). To the best of our knowledge, this is the first report of the occurrence of TYLCV in TN. The virus was detected in a tomato plant grown from seed. The seed transmissibility of TYLCV remains controversial (Perry 2018; and references therein); thus, the most likely source of infection in this report is transmission by rare viruliferous vectors (Bemisia tabaci). It remains unknown, however, whether TYLCV is endemic in TN, or recently introduced by mobile vectors from neighboring states. The presence of TYLCV has been reported in Alabama (Akad et al. 2007), Kentucky (d
{"title":"First Report on the Occurrence of Tomato Yellow Leaf Curl Virus in Tennessee.","authors":"Amanda Penicks, Jakob D Johnson, Annette L Wszelaki, Leslie L Domier, M R Hajimorad","doi":"10.1094/PDIS-07-24-1512-PDN","DOIUrl":"https://doi.org/10.1094/PDIS-07-24-1512-PDN","url":null,"abstract":"<p><p>In the summer of 2021, a field survey of several tomato-growing counties in Tennessee (TN) was conducted for plants exhibiting virus-like symptoms. While scouting in September in Grainger County, one of the largest areas under tomato (Solanum lycopersicum) production in TN, leaves from six tomato plants (cv. BHN 589) growing on a farm located near Rutledge were collected and subsequently stored at -80˚C. Only one of the plants exhibited symptoms typical of tomato yellow leaf curl virus (TYLCV) infection, which included chlorosis, leaf curling, downward cupping, thickening, and mottling. Total DNA was isolated using the DNeasy Plant Mini Kit (Qiagen, Santa Clara, CA) and subjected to PCR using primers TYv2337F (5'-ACGTAGGTCTTGACATCTGTTGAGCTC-3') and TYc138-R: (5'-AAGTGGGTCCCACAATTGCAAGAC-3') and Ex-Taq polymerase (Takara Bio, Mountain View, CA) to amplify a 634-bp genomic fragment of TYLCV (Alkowni et al. 2019). Primers against tomato elongation factor-1 served as internal PCR control (Dias et al. 2023). Each primer set amplified amplicons of expected sizes; however, the TYLCV fragment was detected only from the plant exhibiting typical symptoms of infection. Amplicons were purified with the QIAquick PCR purification kit (Qiagen) and sequenced directly bi-directionally by Eurofins USA using the above primers. The resultant sequences were edited and analyzed with CLC Genomic Workbench v. 24.0.1. Blast analysis of the sequences (606 nts) against those available in GenBank showed 93 TYLCV isolates with over 95% nucleotide sequence identity. Subsequently, the full-length genome was PCR amplified using primers TYBamHIv (5'- GGATCCACTTCTAAATGAATTTCCTG-3') and TYBamHI2c (5'-GGATCCCACATAGTGCAAGACAAAC-3') (Rojas et al. 2007), ligated into pGEM-T (Promega, Madison, WI) and cloned. Plasmids were purified using QIAprep Spin Miniprep kit (Qiagen) and five independent plasmids clones were sequenced using Oxford Nanopore sequencing (v14 library chemistry & R10.4.1 flow cell) by Eurofins USA. The resultant sequences were edited and analyzed with CLC Genomic Workbench and a consensus sequence representing the full-length genome (2,781 nts) was generated and submitted to GenBank (Accession No. PP505780). Blast analysis showed over 98% nucleotide sequence identity with 100 TYLCV isolates from GenBank. The highest sequence identity of 98.6% was with the sequence of an isolate from Florida (AY530931). To the best of our knowledge, this is the first report of the occurrence of TYLCV in TN. The virus was detected in a tomato plant grown from seed. The seed transmissibility of TYLCV remains controversial (Perry 2018; and references therein); thus, the most likely source of infection in this report is transmission by rare viruliferous vectors (Bemisia tabaci). It remains unknown, however, whether TYLCV is endemic in TN, or recently introduced by mobile vectors from neighboring states. The presence of TYLCV has been reported in Alabama (Akad et al. 2007), Kentucky (d","PeriodicalId":20063,"journal":{"name":"Plant disease","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142140788","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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