First report of pearl millet bacterial leaf blight caused by Pantoea stewartii subspecies indologenes in India

Pearl millet (Cenchrus americanus L.) field-grown plants of cv. 7042S shown unusual water-soaked lesions on leaf tips spreading towards the leaf base from Manasagangothri region (12.31°N 76.61°E), Karnataka, a southern Indian state during March 2020. Later those infected plants showed extensive necrosis and typical leaf blight symptoms with 70% disease incidence and 59% severity. Surface sterilized (3 x 3 mm) infected leaf tissues were crushed in 1mL sterile distilled water and streaked onto nutrient agar media. Bright-yellowish, circular, mucoid single bacterial colonies (PPi-M1) with regular margin were recovered after 24 hours of incubation at 28oC, and the same bacterial colonies were used for further biochemical and molecular characterization. The isolate, PPi-M1 found as gram-negative rods, gelatin, starch hydrolysis negative, and catalase, indole production positive. The partial sequence of 16S rRNA gene (primers: 27F/1492R) of the isolate PPi-M1 was amplified, sequenced, and curated sequence submitted to NCBI GenBank (accession number: MN808555). In nucleotide BLAST search for homologous sequences, 99.5% nucleotide matching similarity (1410bp) was observed with other Pantoea stewartii subspecies indologenes strains (MF163274; NR_104928) at NCBI database indicating that our isolate PPi-M1 belongs to this species. In Phylogenetic analysis using the Maximum Likelihood method and Tamura Nei model (1993), PPi-M1 formed a distinct cluster with other Pantoea stewartii strains with bootstrap value >95 and it was distant from P. allii, P. ananatis, P. agglomerans, and P. dispera. Besides, the subspecies-specific PCR assay and subsequent sequencing of galE and recA genes (primers: 3614galE/3614galEc; 3614recA/3614recAc; 372 and 223 bp) also confirmed the identity of the isolate as Pantoea stewartii subspecies indologenes. Further, the pathogenicity test was performed in-planta on 21 days old seedlings of pearl millet cv. CO-10. The bacterial suspension of isolate PPi-M1 (1x108 CFU/ml) was used for inoculation by leaf clipping method (Ke et al. 2017). All the inoculated plants (n=4 leaves per plant; 15 plants) maintained under greenhouse conditions (Temp: 27-29oC; RH: 80-85%) except mock (sterile water inoculation) shown similar water-soaked lesions from the cut end of the leaf, with a definite spreading margin and a typical leaf blight symptom in 8 dpi, as observed in the field. Re-isolated bacterial colonies from infected leaves shared similar morphological characters and molecular identity with inoculated culture, thus proving Koch’s postulates. This pearl millet leaf blight causing bacterial strain PPi-M1 was deposited in the National Agriculturally Important Microbial Culture Collection, Mau, India (accession no.: NAIMCC-B-02508). Previously, P. stewartii was reported to cause leaf blight and rot diseases on rice and maize (Kini et al. 2016; Roper et al. 2011), also the international seed federation has instigated the phytosanitary measures highlighting its true seed transmission ability (Pataky et al. 2003). This study will supplement future pearl millet breeding programs, and to our knowledge, this is the first report of P. s. subsp. indologenes inciting pearl millet leaf blight disease in India.

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First Report of New Bacterial Leaf Blight of Rice Caused by Pantoea ananatis in Southeast China

Plant Disease ◽

10.1094/pdis-05-21-0988-pdn ◽

2021 ◽

Author(s):

Lin Yu ◽

Changdeng Yang ◽

Zhijuan Ji ◽

Yuxiang Zeng ◽

Yan Liang ◽

...

Keyword(s):

Oryza Sativa L ◽

Sequence Similarity ◽

Disease Incidence ◽

Leaf Blight ◽

Bacterial Leaf Blight ◽

Bacterial Suspension ◽

Rrna Gene ◽

Pantoea Ananatis ◽

Southeast China ◽

First Report

In autumn 2020, leaf blight was observed on rice (Oryza sativa L., variety Zhongzao39, Yongyou9, Yongyou12, Yongyou15, Yongyou18, Yongyou1540, Zhongzheyou8, Jiafengyou2, Xiangliangyou900 and Jiyou351) in the fields of 17 towns in Zhejiang and Jiangxi Provinces, China. The disease incidence was 45%-60%. Initially, water-soaked, linear, light brown lesions emerged in the upper blades of the leaves, and then spread down to leaf margins, which ultimately caused leaf curling and blight during the booting-harvest stage (Fig. S1). The disease symptoms were assumed to be caused by Xanthomonas oryzae pv. oryzae (Xoo), the pathogen of rice bacterial blight. 63 isolates were obtained from the collected diseased leaves as previously described (Hou et al. 2020). All isolates showed circular, smooth-margined, yellow colonies when cultured on peptone sugar agar (PSA) medium for 24h at 28℃. The cells were all gram-negative and rod-shaped with three to six peritrichous flagella; positive for catalase, indole, glucose fermentation and citrate utilization, while negative for oxidase, alkaline, phenylalanine deaminase, urease, and nitrate reductase reactions. 16S rRNA gene sequence analysis from the 6 isolates (FY43, JH31, JH99, TZ20, TZ39 and TZ68) revealed that the amplified fragments shared 98% similarity with Pantoea ananatis type strain LMG 2665T (GenBank JFZU01) (Table S3). To further verify P. ananatis identity of these isolates, fragments of three housekeeping genes including gyrB, leuS and rpoB from the 6 isolates were amplified and sequenced, which showed highest homology to LMG 2665T with a sequence similarity of 95%-100% (Table S3). Primers (Brady et al. 2008) and GenBank accession numbers of gene sequences from the 6 isolates are listed in Table S1 and Table S2. Phylogenetic analysis of gyrB, leuS and rpoB concatenated sequences indicated that the 6 isolates were clustered in a stable branch with P. ananatis (Fig. S2). Based on the above morphological, physiological, biochemical and molecular data, the isolates are identified as P. ananatis. For pathogenicity tests, bacterial suspension at 108 CFU/mL was inoculated into flag leaves of rice (cv. Zhongzao39) at the late booting stage using clipping method. Water was used as a negative control. The clipped leaves displayed water-soaked lesions at 3 to 5 days after inoculation (DAI); then the lesion spread downward and turned light brown. At about 14 DAI, blight was shown with similar symptoms to those samples collected from the rice field of Zhejiang and Jiangxi provinces (Fig. S1). In contrast, the control plants remained healthy and symptomless. The same P. ananatis was re-isolated in the inoculated rice plants, fulfilling Koch’s postulates. In the past decade, P. ananatis has been reported to cause grain discoloration in Hangzhou, China (Yan et al. 2010) and induce leaf blight as a companion of Enterobacter asburiae in Sichuan province, China (Xue et al. 2020). Nevertheless, to the best of our knowledge, this is the first report of P. ananatis as the causative agent of rice leaf blight in southeast China. This study raises the alarm that the emerging rice bacterial leaf blight in southeast China might be caused by a new pathogen P. ananatis, instead of Xoo as traditionally assumed. Further, the differences of occurrence, spread and control between two rice bacterial leaf blight diseases caused by P. ananatis and Xoo, respectively need to be determined in the future.

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First Report of Stewart's Wilt of Maize in Argentina Caused by Pantoea stewartii

Plant Disease ◽

10.1094/pdis-07-12-0668-pdn ◽

2012 ◽

Vol 96 (12) ◽

pp. 1819-1819 ◽

Cited By ~ 4

Author(s):

A. G. Albarracín Orio ◽

E. Brücher ◽

M. C. Plazas ◽

P. Sayago ◽

F. Guerra ◽

...

Keyword(s):

Leaf Growth ◽

Leaf Blight ◽

Bacterial Suspension ◽

Post Inoculation ◽

Pathogenicity Test ◽

First Report ◽

Pantoea Stewartii ◽

Maize Plants ◽

Stewart's Wilt ◽

Negative Controls

Stewart's wilt is a serious disease of corn (Zea mays L.) caused by the bacterium Pantoea stewartii subsp. stewartii (Pss). Typical symptoms of infected fields and dent corn are longitudinal streaks with irregular or wavy margins, which are parallel to the veins and may extend the length of the leaf. These pale to green yellow lesions become dry and brown as the disease progresses producing a leaf blight (4). During the growing seasons 2010 to 2011 and 2011 to 2012, symptoms of bacterial leaf blight of corn were observed in central Argentina maize fields, with an incidence of 54% in Córdoba province. To identify the pathogen, leaves from 10 symptomatic maize plants per field were collected from 15 fields covering a representative geographical area. High populations of morphologically uniform bacteria were isolated from leaf tissues by conventional methods using King's medium B agar (2). Ten representative facultatively anaerobic gram-negative, non-fluorescing, non-motile, catalase positive and oxidase negative rod-shaped and yellow-pigmented bacterial isolates were evaluated further. The biochemical profile obtained was: fermentative metabolism, negative indol, acetoin and hydrogen sulfide production, negative gelatin hydrolysis (22°C), positive acid production from D-glucose and lactose, negative gas production from D-glucose, and negative nitrate reduction (1). All the isolates produced a 300-bp band with PCR using the species specific primer pair PST3581/PST3909c (3). The Pss ATCC 8199 and Pseudomonas fluorescens ATCC 13525 strains were used as positive and negative controls for the PCR assays, respectively. The pathogenicity test was performed by stem inoculation of five to ten P2069 YR maize plants (one to two leaf growth stage) grown in growth chamber. Plants were inoculated by syringe with a 107 to 108 cell/ml bacterial suspension and kept in a humid chamber at 25 to 27°C. Plants inoculated with Pss ATCC 8199 or with sterile water were used as positive and negative control treatments, respectively. The development of symptoms similar to those originally found in the field was observed on all the plants inoculated with the different isolates at 7 to 10 days post inoculation. In addition, symptoms on inoculated plants were similar to those observed for the positive control treatment. No symptoms were found on negative controls. Koch's postulates were fulfilled since bacteria isolated from symptomatic tissue had identical characteristics to isolates used to inoculate plants and to the reference Pss strain for biochemical tests and PCR reaction mentioned above. To our knowledge, this is the first report of P. stewartii subsp. stewartii isolated from diseased maize in Argentina. References: (1) J. G. Holt et al. Page 179 in: Bergey's manual of determinative bacteriology. Williams and Wilkins, Baltimore, MD, 1994. (2) OEPP/EPPO. Bulletin OEPP/EPPO Bulletin, 36: 111, 2006. Pantoea stewartii subsp. stewartii diagnostic. (3) A. Wensing et al. Appl. Environ. Microbiol. 76:6248, 2010. (4) D. G. White Page 4 in: Compendium of corn disease. The American Phytopathology Society, 1999.

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First report of Alternaria alternata causing leaf blight on little millet (Panicum sumatrense) in India.

Plant Disease ◽

10.1094/pdis-06-20-1373-pdn ◽

2020 ◽

Author(s):

Boda Praveen ◽

A. Nagaraja ◽

M. K. Prasanna Kumar ◽

Devanna Pramesh ◽

K. B. Palanna ◽

...

Keyword(s):

Crop Yields ◽

Disease Incidence ◽

Leaf Blight ◽

Post Inoculation ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Pcr Assay ◽

Causal Organism ◽

First Report ◽

Little Millet

Little millet (LM) is a minor cereal crop grown in the Indian sub-continent. During October 2018, dark brown, circular to oval necrotic spots surrounded by concentric rings were observed on the upper leaf surface of the LM (cv. VS-13) grown in the fields of the University of Agricultural Sciences, Bengaluru, India (13.0784oN, 77.5793oE). As the disease progressed, infected leaves became blighted. Disease incidence up to 53% was recorded in 3 fields of 0.4-hectare area each. Thirty symptomatic leaves were collected to isolate the associated causal organism. The margins of diseased tissue were cut into 5 × 5-mm pieces, surface-sterilized in 75% ethanol for 45 seconds followed by 1% sodium hypochlorite for 1 min, finally rinsed in sterile distilled water five times and placed on PDA. After 7 days of incubation at 25°C, greyish fungal colonies appeared on PDA. Single-spore isolations were performed to obtain ten isolates. Pure cultures of the fungus initially produced light gray aerial mycelia that later turned to dark grey. All isolates formed obclavate to pyriform conidia measured 22.66-48.97μm long and 6.55-13.79µm wide with 1-3 longitudinal and 2-7 transverse septa with a short beak (2.55-13.26µm) (n=50). Based on the conidial morphology, the fungus was identified as Alternaria sp. Further, the taxonomic identity of all ten isolates was confirmed as A. alternata using species-specific primers (AAF2/AAR3, Konstantinova et al. 2002) in a PCR assay. Later, one of the isolate UASB1 was selected, and its internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (gapdh), major allergen Alt a 1 (Alt a 1), major endo-polygalacturonase (endoPG), OPA10-2, and KOG1058 genes were amplified in PCR (White et al. 1990; Berbee et al. 1999; Woudenberg et al. 2015), and the resultant products were sequenced and deposited in the NCBI GenBank (ITS, MN919390; gapdh, MT637185; Alt a 1, MT882339; endoPG, MT882340; OPA10-2, MT882341; KOG1058, MT882342). Blastn analysis of ITS, gapdh, Alt a 1, endoPG, OPA10-2, KOG1058 gene sequences showed 99.62% (with AF347031), 97.36% (with AY278808), 99.58% (with AY563301), 99.10% (with JQ811978), 99.05% (with KP124632) and 99.23% (with KP125233) respectively, identity with reference strain CBS916.96 of A. alternata, confirming UASB1 isolate to be A. alternata. For pathogenicity assay, conidial suspension of UASB1 isolate was spray inoculated to ten healthy LM (cv. VS-13) plants (45 days old) maintained under protected conditions. The spore suspension was sprayed until runoff on healthy leaves, and ten healthy plants sprayed with sterile water served as controls. Later, all inoculated and control plants were covered with transparent polyethylene bags and were maintained in a greenhouse at 28±2 ◦C and 90% RH. The pathogenicity test was repeated three times. After 8 days post-inoculation, inoculated plants showed leaf blight symptoms as observed in the field, whereas no disease symptoms were observed on non-inoculated plants. Re-isolations were performed from inoculated plants, and the re-isolated pathogen was confirmed as A. alternata based on morphological and PCR assay (Konstantinova et al. 2002). No pathogens were isolated from control plants. There is an increasing acreage of LM crop in India, and this first report indicates the need for further studies on leaf blight management and the disease impacts on crop yields.

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First report of bacterial leaf blight caused by Xanthomonas hortorum pv. carotae on carrots in Spain

Plant Disease ◽

10.1094/pdis-11-20-2493-pdn ◽

2021 ◽

Author(s):

José Luis Palomo Gómez ◽

Maria Shima ◽

Adela Monterde ◽

Inmaculada Navarro ◽

Silvia Barbé ◽

...

Keyword(s):

Sequence Analysis ◽

Xanthomonas Campestris ◽

Negative Control ◽

Leaf Blight ◽

Bacterial Leaf Blight ◽

Pcr Analysis ◽

Bacterial Suspension ◽

Rrna Gene ◽

Distilled Water ◽

First Report

In September 2019, symptoms resembling those of bacterial leaf blight were observed on carrot plants (Daucus carota L. subsp. sativus Hoffm.) cv. Romance cultivated in commercial plots in Chañe (Segovia), Spain. Symptoms were observed in two plots surveyed representing three hectares, with an incidence greater than 90%, and also in some plots in other nearby municipalities sown with the same batch of seeds. The lesions observed at the ends of the leaves were initially yellow that develop dark brown to black with chlorotic halos on leaflets that turned necrotic. Yellow, Xanthomonas-like colonies were isolated onto YPGA medium (Ridé 1969) from leaf lesions. Two bacterial isolates were selected and confirmed by real-time PCR using a specific primer set for Xanthomonas hortorum pv. carotae (Temple et al. 2013). All isolates were gram-negative, aerobic rods positive for catalase, able of hydrolyzing casein and aesculin and growing at 2% NaCl, while were negative for oxidase and urease tests. Sequences of 16S rRNA gene showed 100% similarity with Xanthomonas campestris, X. arboricola, X. gardneri, X. cynarae strains (GenBank accession numbers: MW077507.1 and MW077508.1 for the isolates CRD19-206.3 and CRD19-206.4, respectively). However, the resulting phylogeny of multilocus sequence analysis (MLSA) of a concatenation of the housekeeping genes atpD, dnaK, and efp (Bui Thi Ngoc et al. 2010), by using neighbour-joining trees generated with 500 bootstrap replicates, grouped the two isolates with the X. hortorum pv. carotae M081 strain (Kimbrel et al. 2011) (GenBank accession numbers: MW161270 and MW161271 for atpD for the two isolates, respectively; MW161268 and MW161269 for dnaK; MW161272 and MW161273 for efp). A pairwise identity analysis revealed a 100% identity between all three isolates. Pathogenicity of the isolates was tested by spray inoculation (Christianson et al. 2015) with a bacterial suspension (108 CFU/ml) prepared in sterile distilled water at 3 to 4 true-leaf stage (six plants per isolate). Sterile distilled water was used as negative control. The inoculated plants were incubated in a growth chamber (25°C and 95% relative humidity [RH]) for 72 h, and then transferred to a greenhouse at 24 to 28°C and 65% RH. Characteristic leaf blight symptoms developed on inoculated carrot plants, while no symptoms were observed on the negative control plants 20 days after inoculation. The bacterium was re-isolated from symptomatic tissue and the identity confirmed through PCR analysis. Based on PCR, morphological and phenotypic tests, sequence analysis, and pathogenicity assays, the isolates were identified as X. hortorum pv. carotae. To our knowledge, this is the first report of bacterial leaf blight of carrot caused by X. hortorum pv. carotae in Spain, and the first molecular and pathological characterization. It is important to early detect this pathogen and take suitable measures to prevent its spread, since it could cause yield losses for a locally important crop such as carrot.

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First Report of Leaf Spot Caused by Didymella americana on Cassia nomame in China

Plant Disease ◽

10.1094/pdis-08-21-1799-pdn ◽

2021 ◽

Author(s):

Weiming Sun ◽

Lina Feng ◽

Xiaolei Wen ◽

Bojia Han ◽

Danrun Xing ◽

...

Keyword(s):

Sequence Analysis ◽

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Rrna Gene ◽

Pathogenicity Test ◽

Koch’S Postulates ◽

First Report ◽

Initial Symptoms ◽

Koch's Postulates

Cassia nomame (Sieb.) Kitagawa is an annual plant in the Leguminousae family. The aerial parts of C. nomame have been used as tonic and diuretic in Korea and Japan (Syed et al. 2019). A leaf spot was observed on the leaves of a 1-year-old C. nomame landrace in Changli County (39.42°N, 119.10°E), Qinhuangdao City, Hebei Province during August to October in 2018. In many fields (n≥3), the disease incidence over 80% in the middle and late stage of plant growth. Symptoms on leaves in one field began with many small, dark necrotic spot lesions. Later, the lesions spread to round-to-oval, slightly sunken in the center, and large necrotic patches with indefinite margins. Finally, lesions coalesced and resulted in defoliation. Lesions were occasionally observed on the pods. Symptoms on the pods were initially small, dark spots and then expanded to large necrotic patches with irregular edges. Symptomatic tissues (n=32) from pods and leaves were cut into 3 to 8 mm2 squares, surface disinfested with 75% ethanol for 10 s, rinsed with sterile distilled water, then placed on potato dextrose agar (PDA) at 28℃. After 3 days, ten isolates with consistent characteristics were obtained with a frequency 52.6%. The isolates on PDA were round, initially pale and had little aerial mycelium, gradually turned olive green and had dense wool-like dense aerial mycelia after 3 days. Conidia were hyaline, smooth, solitary, and elliptical. The conidia measured 5.4 to 8.2 μm × 2.5 to 3.8 μm (n=50), and has two oil bodies positioning at opposite poles. Pigmented chlamydospores were spherical or nearly pear-shaped, and solitary. Black fructifications (pycnidia) were produced profusely on PDA after subculture for 3 days. All the isolates were similar to Didymella sp. in morphology (Aveskamp et al. 2009). Choice three isolates YSGUO8 YSGGUO8-a and YSGGUO8-b to be further characterized by sequencing of the internal transcribed spacer (ITS), actin gene, and 28S large subunit of the nuclear rRNA gene (LSU) (Zhang et al. 2017). The sequences of three strains (MK836417 MZ484072 and MZ484073 for ITS, MK837604 MZ593675 and MZ593676 for actin, MK843781 MZ836208 and MZ836207 for LSU, respectively) showed 99% to 100% similarity with Didymella americana K-004 (KY070279 for ITS,KY070285 for LSU), Phoma americana CBS 256.65 (FJ426973 for ITS, FJ426871 for actin, MH870196 for LSU) and P. americana CBS 185.85 (FJ426972 for ITS, FJ426870 for actin, GU237990 for LSU) in GenBank. The fungi were identified as D. americana (formerly P. americana or Peyronellaea americana) on the basis of morphological characteristics and sequence analysis. A pathogenicity test was conducted with three times on 1-year-old C. nomame strain at the 4 to 6 compound leaf stage. Conidia were obtained from 7-day-old PDA cultures grown at 28℃ with a 12-h photoperiod. Koch’s postulates were fulfilled by spray inoculating ten healthy young plants with 106 conidia per milliliter of D. americana strain YSGUO8, and sterile water as the control. After inoculation, the plants were managed at 28℃, 60% relative humidity and a 12-h photoperiod. After 5 to 8 days, the inoculated leaves developed small and dark spots lesions similar to those observed on the leaves with initial symptoms in the field. The control leaves remained symptomless. The same fungi were re-isolated from infected leaves by morphology observation and sequence analysis, confirming Koch's postulates. D. americana has caused leaves spot on Table Beet in New York (Vaghefi et al. 2016). To our knowledge, this is the first report of D. americana causing leaf spot of C. nomame in China.

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First Report of Bleeding Canker of Pear Tree Trunks Caused by Dickeya fangzhongdai in Korea

Plant Disease ◽

10.1094/pdis-09-20-1948-pdn ◽

2021 ◽

Author(s):

Eu Ddeum Choi ◽

Youngmin Kim ◽

Yerim Lee ◽

Min-Hye Jeong ◽

Gyoung Hee Kim ◽

...

Keyword(s):

16S Rrna ◽

Intergenic Spacer ◽

Fine Powder ◽

Luria Bertani ◽

Bacterial Suspension ◽

Post Inoculation ◽

Pathogenicity Test ◽

Flower Bud ◽

First Report ◽

Pear Tree

Pears (Pyrus pylifolia L.) are cultivated nationwide as one of the most economically important fruit trees in Korea. At the end of October 2019, bleeding canker was observed in a pear orchard located in Naju, Jeonnam Province (34°53′50.54″ N, 126°39′00.32″ E). The canker was observed on trunks and branches of two 25-year-old trees, and the diseased trunks and branches displayed partial die-back or complete death. When the bark was peeled off from the diseased trunks or branches, brown spots or red streaks were found in the trees. Bacterial ooze showed a rusty color and the lesion was sap-filled with a yeasty smell. Trunks displaying bleeding symptoms were collected from two trees. Infected bark tissues (3 × 3 mm) from the samples were immersed in 70% ethanol for 1 minute, rinsed three times in sterilized water, ground to fine powder using a mortar and pestle, and suspended in sterilized water. After streaking each suspension on Luria-Bertani (LB) agar, the plates were incubated at 25°C without light for 2 days. Small yellow-white bacterial colonies with irregular margins were predominantly obtained from all the samples. Three representative isolates (ECM-1, ECM-2 and ECM-3) were subjected to further characterization. These isolates were cultivated at 39 C, and utilized (-)-D-arabinose, (+) melibiose, (+)raffinose, mannitol and myo-inositol but not 5-keto-D-gluconate, -gentiobiose, or casein. These isolates were identified as Dickeya sp. based on the sequence of 16S rRNA (MT820458-820460) gene amplified using primers 27f and 1492r (Heuer et al. 2000). The 16S rRNA sequences matched with D. fangzhongdai strain ND14b (99.93%; CP009460.1) and D. fangzhongdai strain PA1(99.86%; CP020872.1). The recA, fusA, gapA, purA, rplB, and dnaX genes and the intergenic spacer (IGS) regions were also sequenced as described in Van der wolf et al. (2014). The recA (MT820437-820439), fusA (MT820440-820442), gapA (MT820443-820445), purA (MT820446-820448), rplB (MT820449-820451), dnaX (MT820452-820454) and IGS (MT820455-820457) sequences matched with D. fangzhongdai strains JS5, LN1 and QZH3 (KT992693-992695, KT992697-992699, KT992701-992703, KT992705-992707, KT992709-992711, KT992713-992715, and KT992717-992719, respectively). A neighbor-joining phylogenetic analysis based on the concatenated recA, fusA, gapA, purA, rplB, dnaX and IGS sequences placed the representative isolates within a clade comprising D. fangzhongdai. ECM-1 to 3 were grouped into a clade with one strain isolated from waterfall, D. fangzhongdai ND14b from Malaysia. Pathogenicity test was performed using isolate ECM-1. Three two-year-old branches and flower buds on 10-year-old pear tree (cv. Nittaka), grown at the National Institute of Horticultural and Herbal Science Pear Research Institute (Naju, Jeonnam Province in Korea), were inoculated with 10 μl and 2 μl of a bacterial suspension (108 cfu/ml), respectively, after wounding inoculation site with a sterile scalpel (for branch) or injecting with syringe (for flower bud). Control plants were inoculated with water. Inoculated branches and buds in a plastic bag were placed in a 30℃ incubator without light for 2 days (Chen et al. 2020). Both colorless and transparent bacterial ooze and typical bleeding canker were observed on both branches and buds at 3 and 2 weeks post inoculation, respectively. No symptoms were observed on control branches and buds. This pathogenicity assay was conducted three times. We reisolated three colonies from samples displaying the typical symptoms and checked the identity of one by sequencing the dnaX locus. Dickeya fangzhongdai has been reported to cause bleeding canker on pears in China (Tian et al. 2016; Chen et al. 2020). This study will contribute to facilitate identification and control strategies of this disease in Korea. This is the first report of D. fangzhongdai causing bleeding canker on pears in Korea.

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First Report of Phytophthora drechsleri Associated with Stem and Foliar Blight of Gynura bicolor in Taiwan

Plant Disease ◽

10.1094/pdis-12-10-0931 ◽

2011 ◽

Vol 95 (7) ◽

pp. 874-874 ◽

Cited By ~ 1

Author(s):

Y. M. Shen ◽

C. H. Chao ◽

H. L. Liu

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

Hyphal Growth ◽

Pathogenicity Test ◽

Distilled Water ◽

First Report ◽

Foliar Blight ◽

Dual Cultures ◽

Phytophthora Drechsleri ◽

Gynura Bicolor

Gynura bicolor (Roxb. ex Willd.) DC., known as Okinawa spinach or hong-feng-cai, is a commonly consumed vegetable in Asian countries. In May 2010, plants with blight and wilt symptoms were observed in commercial vegetable farms in Changhua, Taiwan. Light brown-to-black blight lesions developed from the top of the stems to the petioles and extended to the base of the leaves. Severely infected plants declined and eventually died. Disease incidence was approximately 20%. Samples of symptomatic tissues were surface sterilized in 0.6% NaOCl and plated on water agar. A Phytophthora sp. was consistently isolated and further plated on 10% unclarified V8 juice agar, with daily radial growths of 7.6, 8.6, 5.7, and 2.4 mm at 25, 30, 35, and 37°C, respectively. Four replicates were measured for each temperature. No hyphal growth was observed at 39°C. Intercalary hyphal swellings and proliferating sporangia were produced in culture plates flooded with sterile distilled water. Sporangia were nonpapillate, obpyriform to ellipsoid, base tapered or rounded, and 43.3 (27.5 to 59.3) × 27.6 (18.5 to 36.3) μm. Clamydospores and oospores were not observed. Oospores were present in dual cultures with an isolate of P. nicotianae (p731) (1) A2 mating type, indicating that the isolate was heterothallic. A portion of the internal transcribed spacer sequence was deposited in GenBank (Accession No. HQ717146). The sequence was 99% identical to that of P. drechsleri SCRP232 (ATCC46724) (3), a type isolate of the species. The pathogen was identified as P. drechsleri Tucker based on temperature growth, morphological characteristics, and ITS sequence homology (3). To evaluate pathogenicity, the isolated P. drechsleri was inoculated on greenhouse-potted G. bicolor plants. Inoculum was obtained by grinding two dishes of the pathogen cultured on potato dextrose agar (PDA) with sterile distilled water in a blender. After filtering through a gauze layer, the filtrate was aliquoted to 240 ml. The inoculum (approximately 180 sporangia/ml) was sprayed on 24 plants of G. bicolor. An equal number of plants treated with sterile PDA processed in the same way served as controls. After 1 week, incubation at an average temperature of 29°C, blight and wilt symptoms similar to those observed in the fields appeared on 12 inoculated plants. The pathogen was reisolated from the lesions of diseased stems and leaves, fulfilling Koch's postulates. The controls remained symptomless. The pathogenicity test was repeated once with similar results. G. bicolor in Taiwan has been recorded to be infected by P. cryptogea (1,2), a species that resembles P. drechsleri. The recorded isolates of P. cryptogea did not have a maximal growth temperature at or above 35°C (1,2), a distinctive characteristic to discriminate between the two species (3). To our knowledge, this is the first report of P. drechsleri being associated with stem and foliar blight of G. bicolor. References: (1) P. J. Ann. Plant Pathol. Bull. 5:146, 1996. (2) H. H. Ho et al. The Genus Phytophthora in Taiwan. Institute of Botany, Academia Sinica, Taipei, 1995. (3) R. Mostowfizadeh-Ghalamfarsa et al. Fungal Biol. 114:325, 2010.

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First Report of Fusarium pernambucanum Causing Fruit Rot of Muskmelon in China

Plant Disease ◽

10.1094/pdis-07-21-1520-pdn ◽

2021 ◽

Author(s):

Xianping Zhang ◽

Jiwen Xia ◽

Jiakui Liu ◽

Dan Zhao ◽

Lingguang Kong ◽

...

Keyword(s):

Phylogenetic Analyses ◽

Apical Cell ◽

Morphological Characteristics ◽

Fruit Rot ◽

Likelihood Method ◽

Correct Identification ◽

Pathogenicity Test ◽

First Report ◽

The Core ◽

Representative Isolate

Muskmelon (Cucumis melo L.) is one of the most widely cultivated and economically important fruit crops in the world. However, many pathogens can cause decay of muskmelons; among them, Fusarium spp. is the most important pathogen, affecting fruit yield and quality (Wang et al. 2011). In May 2017, fruit rot symptoms were observed on ripening muskmelons (cv. Jipin Zaoxue) in several fields in Liaocheng of Shandong Province, China. Symptoms appeared as brown, water-soaked lesions, irregularly circular in shape, with the lesion size ranging from a small spot (1 to 2 cm) to the decay of the entire fruit. The core and the surface of the infected fruit were covered with white to rose-reddish mycelium. Two infected muskmelons were collected from each of two fields, 10 km apart. Tissues from the inside of the infected fruit were surface disinfected with 75% ethanol for 30 s, and cultured on potato dextrose agar (PDA) at 25 °C in the dark for 5 days. Four purified cultures were obtained using the single spore method. On carnation leaf agar (CLA), macroconidia had a pronounced dorsiventral curvature, falcate, 3 to 5 septa, with tapered apical cell, and foot-shaped basal cell, measuring 19 to 36 × 4 to 6 μm. Chlamydospores were abundant, 5.5–7.5 μm wide, and 5.5–10.5 μm long, ellipsoidal or subglobose. No microconidia were observed. These morphological characteristics were consistent with the descriptions of F. pernambucanum (Santos et al. 2019). Because these isolates had similar morphology, one representative isolate was selected for multilocus phylogenetic analyses. DNA was extracted from the representative isolate using the CTAB method. The nucleotide sequences of the internal transcribed spacers (ITS) (White et al. 1990), translation elongation factor 1-α gene (TEF1), RNA polymerase II second largest subunit gene (RPB2), calmodulin (CAM) (Xia et al. 2019) were amplified using specific primers, sequenced, and deposited in GenBank (MN822926, MN856619, MN856620, and MN865126). Based on the combined dataset of ITS, TEF1, RPB2, CAM, alignments were made using MAFFT v. 7, and phylogenetic analyses were processed in MEGA v. 7.0 using the maximum likelihood method. The studied isolate (XP1) clustered together with F. pernambucanum reference strain URM 7559 (99% bootstrap). To perform pathogenicity test, 10 μl of spore suspensions (1 × 106 conidia/ml) were injected into each muskmelon fruit using a syringe, and the control fruit was inoculated with 10 μl of sterile distilled water. There were ten replicated fruits for each treatment. The test was repeated three times. After 7 days at 25 °C, the interior of the inoculated muskmelons begun to rot, and the rot lesion was expanded from the core towards the surface of the fruit, then white mycelium produced on the surface. The same fungus was re-isolated from the infected tissues and confirmed to fulfill the Koch’s postulates. No symptoms were observed on the control muskmelons. To our knowledge, this is the first report of F. pernambucanum causing of fruit rot of muskmelon in China. Considering the economic value of the muskmelon crop, correct identification can help farmers select appropriate field management measures for control of this disease.

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First Report of Curvularia aeria on Etlingera linguiformis from Nagaland, India

Plant Disease ◽

10.1094/pdis-01-14-0060-pdn ◽

2014 ◽

Vol 98 (11) ◽

pp. 1580-1580 ◽

Cited By ~ 2

Author(s):

C. Kithan ◽

L. Daiho

Keyword(s):

Leaf Surface ◽

Agricultural Research ◽

Disease Incidence ◽

Indian Agricultural Research Institute ◽

Mountain Range ◽

Pathogenicity Test ◽

Distilled Water ◽

Conidial Suspension ◽

First Report ◽

Initial Symptoms

Etlingera linguiformis (Roxb.) R.M.Sm. of Zingiberaceae family is an important indigenous medicinal and aromatic plant of Nagaland, India, that grows well in warm climates with loamy soil rich in humus (1). The plant rhizome has medicinal benefits in treating sore throats, stomachache, rheumatism, and respiratory complaints, while its essential oil is used in perfumery. A severe disease incidence of leaf blight was observed on the foliar portion of E. linguiformis at the Patkai mountain range of northeast India in September 2012. Initial symptoms of the disease are small brown water soaked flecks appearing on the upper leaf surface with diameter ranging from 0.5 to 3 cm, which later coalesced to form dark brown lesions with a well-defined border. Lesions often merged to form large necrotic areas, covering more than 90% of the leaf surface, which contributed to plant death. The disease significantly reduces the number of functional leaves. As disease progresses, stems and rhizomes were also affected, reducing quality and yield. The diseased leaf tissues were surface sterilized with 0.2% sodium hypochlorite for 2 min followed by rinsing in sterile distilled water and transferred into potato dextrose agar (PDA) medium. After 3 days, the growing tips of the mycelium were transferred to PDA slants and incubated at 25 ± 2°C until conidia formation. Fungal colonies on PDA were dark gray to dark brown, usually zonate; stromata regularly and abundantly formed in culture. Conidia were straight to curved, ellipsoidal, 3-septate, rarely 4-septate, middle cells broad and darker than other two end cells, middle septum not median, smooth, 18 to 32 × 8 to 16 μm (mean 25.15 × 12.10 μm). Conidiophores were terminal and lateral on hyphae and stromata, simple or branched, straight or flexuous, often geniculate, septate, pale brown to brown, smooth, and up to 800 μm thick (2,3). Pathogen identification was performed by the Indian Type Culture Collection, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi (ITCC Accession No. 7895.10). Further molecular identity of the pathogen was confirmed as Curvularia aeria by PCR amplification and sequencing of the internal transcribed spacer (ITS) regions of the ribosomal DNA by using primers ITS4 and ITS5 (4). The sequence was submitted to GenBank (Accession No. MTCC11875). BLAST analysis of the fungal sequence showed 100% nucleotide similarity with Cochliobolus lunatus and Curvularia aeria. Pathogenicity tests were performed by spraying with an aqueous conidial suspension (1 × 106 conidia /ml) on leaves of three healthy Etlingera plants. Three plants sprayed with sterile distilled water served as controls. The first foliar lesions developed on leaves 7 days after inoculation and after 10 to 12 days, 80% of the leaves were severely infected. Control plants remained healthy. The inoculated leaves developed similar blight symptoms to those observed on naturally infected leaves. C. aeria was re-isolated from the inoculated leaves, thus fulfilling Koch's postulates. The pathogenicity test was repeated twice. To our knowledge, this is the first report of the presence of C. aeria on E. linguiformis. References: (1) M. H. Arafat et al. Pharm. J. 16:33, 2013. (2) M. B. Ellis. Dematiaceous Hyphomycetes. CMI, Kew, Surrey, UK, 1971. (3) K. J. Martin and P. T. Rygiewicz. BMC Microbiol. 5:28, 2005. (4) C. V. Suberamanian. Proc. Indian Acad. Sci. 38:27, 1955.

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