First Report of Leaf Blight Wilt on Dracaena sanderiana by Pantoea stewartii subsp. indologenes in China

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

Plant Disease ◽

10.1094/pdis-03-21-0669-pdn ◽

2021 ◽

Author(s):

Ashajyothi Mushineni ◽

A Balamurugan ◽

Shashikumara P ◽

Neha Pandey ◽

Dinesh Kumar Agarwal ◽

...

Keyword(s):

Pearl Millet ◽

Disease Incidence ◽

Leaf Blight ◽

Likelihood Method ◽

Bacterial Suspension ◽

Rrna Gene ◽

Microbial Culture ◽

Pathogenicity Test ◽

First Report ◽

Pantoea Stewartii

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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