scholarly journals First report of stalk bacterial soft rot of sugarcane caused by Dickeya zeae in China

Plant Disease ◽  
2020 ◽  
Author(s):  
yanchang Yang ◽  
Ziting Yao ◽  
Mu-Qing Zhang ◽  
Chengwu Zou ◽  
Baoshan Chen
Keyword(s):  
Pure Water ◽  
Bacterial Species ◽  
Evolutionary Relationship ◽  
Soft Rot ◽  
Bacterial Suspension ◽  
Rrna Gene ◽  
Post Inoculation ◽  
Distilled Water ◽  
Sugarcane Cultivar ◽  
First Report

In late September 2019, seven stalks of about 1400 stalks of sugarcane cultivar Zhongzhe 1 exhibited soft rot symptoms in a trial plot in Beihai city, Guangxi province of China. Symptoms included scorched and collapsed leaves, maceration of stalks, and sour smelling exudates from the stalks (Supplementary Fig. S1). Severely diseased stalks had collapsed and were dead. Internal stalk fragments of 5 × 5 mm were collected at the junction of healthy and diseased tissue after surface-sterilization of stalks with 70% ethanol for one minute, and three times rinsing with sterile distilled water. Stalk fragments were placed on Luria–Bertani agar medium (1 % w/v tryptone, 0.5 % w/v yeast extract, 1 % w/v NaCl, 1 % w/v agar, pH7.0) and plates were put in an incubator at 30°C for 48h. Four types of bacterial colonies were obtained, and small and white colonies with irregular margins were the most dominant. A single colony of each type was diluted in sterile distilled water and aliquots of each suspension were streaked on fresh medium plates to obtain pure cultures. Ten eight-week-old stalks (11 th leaf stage) of sugarcane plants, which derived from cuttings of symptomless cultivar Zhongzhe 1, were inoculated by injection of 300 μl of bacterial suspension (3.5x108 CFU/ml) into the stalks. Another 10 stalks were injected with pure water and served as control. The inoculated plants were kept in a greenhouse at 25-37℃.Among the four types of bacteria, only strain BH9 induced symptoms that were identical to those of diseased canes observed in the field (Supplementary Fig. S1). Elongated water-soaked lesions were observed around the inoculation sites three days post inoculation. Five of the 10 BH9-inoculated plants had collapsed two days later. Water-soaked stalks had a sour smell similar to the filed diseased plants. Eight days post inoculation, all BH9-inoculated plants exhibited symptoms but control plants remained symptomless up to 30 days after inoculation. Uniform white colonies with irregular margins were isolated from the inoculated stalks that developed soft rot symptom, and these bacteria caused again stalk soft rot symptoms when inoculated to a new batch of 10 healthy plants. The 16S rRNA gene of strain BH9 was amplified by PCR with primer pair fD2/rP1 and the PCR amplicons from three independent colonies were sequenced. The sequences of the three amplicons were identical (Accession No. MT723897). BLAST alignments of the 16S rDNA sequence from BH9 strain with the GenBank database revealed that BH9 belonged to the genus Dickeya (98.5% identity between D. zeae BH9 and D. zeae EC1). Further PCR assays and sequencing of three genes, DNA polymerase III gamma subunit gene dnaX with primers dnaXf/dnaXr, DNA gyrase gene gyrB with primers gyrBf1/gyrBr1, and recombinase A gene recA with primers recAf/recAr, were performed to identify the species within the genus Dickeya (Zhang et al., 2014). BH9 sequences of these genes (Accession No. MT723898 to MT723900) had highest identity (97.5%, 97.6%, and 97.7%, respectively) with those from D. zeae EC1 (GenBank accession No. CP006929.1). To determine the evolutionary relationship of BH9 to other Dickeya species and strains, a phylogenetic analysis was performed using dnaX, gyrB, and recA sequences. As shown in Supplementary Fig. S2, BH9 clustered with D. zeae strains and formed a lineage distinguishable from other Dickeya species. Among the closest strains, D. zeae NCPPB3531 (Accession No. CM001980.1) was isolated from potato and D. zeae CSL RW192 (Accession No. CM001972.1) from river water (Pritchard et al., 2013). Consequently, strain BH9 was identified as D. zeae. This bacterial species has been reported to cause soft rot in rice (Pu et al., 2012), banana (Zhang et al., 2014), maize (Martinez-Cisneros et al., 2014), and clivia (Hu et al., 2018). To the best of our knowledge, this is the first report of a bacterial stalk rot caused by D. Zeae in sugarcane. In fact, low incidence of D. zeae-caused stalk soft rot was recently found in sugarcane fields in Fusui County, about 150 km north to Beihai. Given the potential threat of this disease to the local sugarcane industry, the mode of transmission, cultivar resistance, and measures to control the disease should be investigated.

scholarly journals First Report in China of Soft Rot of Ginger Caused by Pythium aphanidermatum

Plant Disease ◽  
2014 ◽  
Vol 98 (7) ◽  
pp. 1011-1011 ◽  
Author(s):  
Y. Li ◽  
L. G. Mao ◽  
D. D. Yan ◽  
X. M. Liu ◽  
T. T. Ma ◽  
...  
Keyword(s):  
Aerial Mycelium ◽  
Zingiber Officinale ◽  
Average Diameter ◽  
Soft Rot ◽  
Pythium Aphanidermatum ◽  
Selective Medium ◽  
Rrna Gene ◽  
Distilled Water ◽  
First Report ◽  
Pythium Soft Rot

Ginger (Zingiber officinale Roscoe) is an important commercial crop planted on more than 13,000 ha annually in Anqiu city, Shandong Province, China. From 2010 to 2011, the incidence of Pythium soft rot disease on cv. Laiwu Big Ginger reached 40 to 75% in Anqiu and yield losses of up to 60% were observed. The disease symptoms included brown spots on ginger rhizomes followed by soft rot, stems and leaves above ground becoming withered and yellow, and water soaking on the collar region. The soft rot did not produce offensive odors, which is different from bacterial rots (2). Forty symptomatic rhizomes were sampled from eight farms. Martin's method (1) was used to isolate the pathogen. Ten pieces from each rhizome were washed with sterile distilled water for 30 s and plated on Martin's selective medium at 26°C in a chamber without light. Colonies grew with cottony aerial mycelium. Main hyphae were 5.7 to 9.6 μm wide. Globose sporangia consisting of terminal complexes of swollen hyphal branches were 11.4 to 18.3 μm wide. The average diameter of zoospores was 9.2 μm. The oogonia were globose and smooth, with a diameter of 21 to 33 μm. The sequences of the rRNA gene internal transcribed spacer (ITS) regions 1 and 2 and the 5.8S gene of five isolates were amplified using primers ITS1 and ITS4 (4), and the nucleotide sequence was the same as isolate No. 2, which was deposited in GenBank (Accession No. KC594034). A BLAST search showed 99% identity with Pythium aphanidermatum strain 11-R-8 (Accession No. JQ898455.1). Pathogenicity tests of five isolates were carried out in a greenhouse. Sixty plants (cv. Laiwu Big Ginger) were grown for 30 days in plastic pots (diameter 20 cm) in sandy soil (pH 5.48) and inoculated. Ten plants were used as untreated controls. Five isolates were grown on Martin's liquid medium for 72 h and the spores were harvested in sterile distilled water. Aqueous spore suspensions of the five isolates were adjusted with deionized water to 1 × 108 CFU/ml and injected with a syringe into the soil around the rhizome of the plants. Plants were then placed in the greenhouse at 24 to 26°C and assessed for rhizome rot on the 14th day after inoculation. The inoculated isolates were recovered from the diseased rhizomes, confirming their pathogenicity. To our knowledge, this is the first report of ginger Pythium soft rot caused by P. aphanidermatum in China. Ginger Pythium soft rot caused by P. myriotylum is reported in Taiwan (3). References: (1) F. N. Martin. Page 39 in: The Genus Pythium. American Phytopathological Society, St. Paul, MN, 1992. (2) E. E. Trujillo. Diseases of Ginger (Zingiber officinale) in Hawaii, Circular 62, Hawaii Agricultural Experiment Station, University of Hawaii, December 1964. (3) P. H. Wang. Lett. Appl. Microbiol. 36:116, 2003. (4) T. J. White. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

scholarly journals First Report of Pseudomonas oryzihabitans Causing Stem and Leaf Rot on Muskmelon in China

Plant Disease ◽  
2021 ◽  
Author(s):  
JuFen Li ◽  
Ganghan Zhou ◽  
Tan Wang ◽  
Tao Lin ◽  
yiwen wang ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Suspension ◽  
Rrna Gene ◽  
Distilled Water ◽  
Phylogenetic Tree Analysis ◽  
First Report ◽  
Rot Disease ◽  
The 16S Rrna Gene ◽  
Leaf Rot

Muskmelon (Cucumis melo L.) is an important economic crop in China, which is planted on more than 376, 000 hectares with over 13 million tons of annual fruit production. In February 2020, a serious bacterial stem and leaf rot disease on muskmelon plants was observed in greenhouses in Liguo Town, Ledong County, Hainan Province, China (18.54° N, 108.87° E), with disease incidences being approximately 10 to 12%. Initially, soft rot symptoms appeared on petioles and stems, showing yellow bacterial ooze signs, which was different from the milky white ooze produced by Erwinia tracheiphila infection. The infected tissues of petioles, stems, and leaves eventually developed into browning and withering symptoms. To isolate and identify the causal agent, the lesion tissues were sterilized by immersion in 75% ethanol for 30 s, washed three times with sterile water, and then cut and soaked in 1 ml of distilled water for 10 min. The suspension was serially diluted and spread on Luria-Bertani agar (LB) medium. After incubation at 28°C for 24 to 36 h, the resulted bacterial colonies were tiny and were streaked on LB plate for further culture. After purification, the colonies were yellow, circular, smooth-margined, and two independent representative isolates CM-11 and CM-12 were used for further validation experiments. The electron microscope analysis showed that the pathogen was rod-shaped, with a length of 1.34 ± 0.22 μm and a width of 0.54 ± 0.06 μm (N=50), and had a single terminal flagellum. The gram staining of the two isolates was negative. Moreover, the tested strains were positive for catalase but negative for oxidase and were able to utilize D-glucose, L-arabinose, and D-mannitol. Morphological, physiological, and biochemical characteristics of both isolates were consistent with those of Pseudomonas spp. To verify the species identity of the bacterial pathogens, genomic DNA of isolates CM-11 and CM-12 was extracted and several conserved genes were amplified and sequenced, including the 16S rRNA gene with primers 27F/1492R (GenBank MW187499 and MW187500), rpoB gene with primers V4/LAPS27 (MW201910 and MW446819), and gyrB gene with primers gyrBBAUP2/APrU (MW187501 and MW187502) (Mulet et al. 2010). In the BLAST analysis, the 16S rRNA sequences showed a 99% similarity to that of Pseudomonas oryzihabitans strains TH19 (LC026009), AA21 (MG571765). The rpoB and gyrB sequences showed high similarity (> 98%) to P. oryzihabitans strains FDAARGOS_657. The phylogenetic tree analysis of rpoB and gyrB genes further verified that the two isolates CM-11 and CM-12 were most closely related to P. oryzihabitans species. Consequently, the two pathogenic isolates CM-11 and CM-12 were identified as P. oryzihabitans. Both strains of CM-11 and CM-12 were tested to accomplish Koch's postulates. Young branches of muskmelons (cultivar Yugu, 10 days after pollination) were chosen as the material for inoculation. Ten healthy detached branches were placed in 15 ml tubes by submerging the cutting wound in 5 ml of the bacterial suspension (108 CFU/ml). Ten additional branches were implemented with sterilized distilled water as a negative control. The inoculated branches were placed in a plastic box containing moistened paper at 28°C. Rotting symptoms appeared within 5 days after infection, while the control samples remained healthy. Bacteria were re-isolated from diseased tissues, and the 16S rRNA gene sequences of the isolates showed the same as those from the original pathogen. Panicle blight and grain discoloration disease caused by P. oryzihabitans on rice has been described in China (Hou et al. 2020). It’s also recently found that P. oryzihabitans caused center blackening disease on muskmelon fruit in Korea (Choi et al. 2019). This study indicated that it was a causative agent of stem and leaf rot disease during the field growth period. To the best of our knowledge, this is the first report of P. oryzihabitans causing muskmelon stem rot in China.

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

Plant Disease ◽  
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.

scholarly journals First Report of Pectobacterium punjabense causing potato soft rot and blackleg in Serbia

Plant Disease ◽  
2021 ◽  
Author(s):  
Marta Loc ◽  
Dragana Milošević ◽  
Maja Ignjatov ◽  
Žarko Ivanović ◽  
Dragana Budakov ◽  
...  
Keyword(s):  
16S Rdna ◽  
Potato Plant ◽  
Soft Rot ◽  
Economic Losses ◽  
Bacterial Suspension ◽  
Distilled Water ◽  
Potato Tubers ◽  
First Report ◽  
Potato Plants ◽  
16S Rdna Pcr

Soft rot and blackleg are common diseases affecting potato (Solanum tuberosum) production in Serbia. Pectinolytic plant pathogens belonging to the genera Pectobacterium cause soft rot and wilt diseases by plant cell wall degradation. These opportunistic phytopathogens lead to considerable economic losses in many potato-growing regions worldwide and are listed among top 10 plant pathogenic bacteria (Mansfield et al. 2012). Potato plants (cv. VR808) with symptoms of wilting, slow growth, stem blackening and tubers softening, were collected from a commercial potato field in Zobnatica (Serbia) in July 2019 and subjected to analysis. All symptoms occurred in the same field and the incidence of symptomatic plants was approximately 5%. Isolation was performed from 10 randomly chosen potato plant and tuber samples, expressing wilting and soft rot symptoms. Plant tissue was surface-disinfected and 1 cm length sections from the margins of lesions were macerated in sterile distilled water for 25 min and streaked on nutrient-agar medium. After 48 h of incubation at 26°C, predominant shiny, cream-colored, round colonies were obtained from all samples. Three representative isolates (MMZKVR1, MMZCVR2, and MMZKVR3) from independent samples were selected randomly and subjected to biochemical and pathogenicity tests. Isolates were gram-negative, nonfluorescent facultative anaerobes, exhibiting pectinolytic activity on potato tuber slices and hypersensitive response on tobacco leaves. They expressed catalase activity but did not express oxidase or acid phosphatase activity or produce indole. All strains grew at 37°C, in 5% NaCl, and reduced nitrate. Pathogenicity of the obtained isolates was tested on 3-week-old healthy potato plants (cv. VR808 and cv. Kiebitz) grown in commercial Baltic Tray Substrate (Hawita) in the greenhouse, as well as on potato tubers of the same varieties. Three potato plant stems per isolate were inoculated by the toothpick piercing method (Duarte et al. 2004) using bacterial suspension (approx. 1 × 108 CFU/ml). Inoculated plants were incubated under plastic bags in a greenhouse at 25 ± 2°C. Blackleg symptoms and stem wilting developed 48 hours after inoculation. No symptoms were observed on plants inoculated with sterile toothpicks dipped in sterile distilled water. The pathogen was re-isolated from symptomatic plants, fulfilling Koch's postulates and sequencing of 16S rDNA confirmed the originally isolated pathogen. Three potato tubers per isolate were inoculated by toothpicks dipped in bacterial suspension (approx. 1 × 108 CFU/ml). Inoculated tubers were placed in a sealed plastic container at 25 ± 2°C. Treatment with sterile distilled water was used as a negative control. Softening of the tissue around the inoculation point developed within 48 h from inoculation, and no symptoms developed on the control tubers. For molecular analyses, total DNA of the isolates was extracted using the DNeasy Plant Mini Kit (Qiagen). The isolates were not detected in diagnostic PCR assays using specific primers Br1F/L1R for the detection of P. brasiliense (Duarte et al. 2004) and primers EXPCCF/EXPCCR for P. catotovorum subsp. carotovorum (Kang et al. 2003). The 16S rDNA PCR amplification was performed using the universal PCR primer pair 27F/1492R (Fredriksson et al. 2013) and followed by Sanger sequencing (Macrogen Europe BV). The BLASTn analysis of sequences (GenBank Accession Numbers MZ048661, MZ048662, and MZ157274) revealed 100% query coverage and 100% identity to the sequences of Pectobacterium punjabense in NCBI (MT242589 and CP038498) isolated from potato in China and Pakistan (Sarfraz et al. 2018), respectively. All three obtained isolates were proposed to belong to Pectobacterium punjabense sp. nov. To further validate the identification, isolate MMZCVR2 of P. punjabense was selected for multilocus sequence analyses of 5 housekeeping genes (gyrA, recA, recN, rpoA and rpoS). The gyrA (MZ161817), recA (MZ161818), recN (MZ161819), rpoA (MZ161820) and rpoS (MZ161821) sequence analysis showed the highest nucleotide identity (99.44 to 100%) with P. punjabense strain SS95 (Sarfraz et al. 2018) previously deposited in NCBI GenBank database. To our knowledge, this is the first report of blackleg and soft rot caused by P. punjabense on potato in Serbia. Pectobacterium punjabense is a newly described species causing soft rot and blackleg disease in potato plants (Sarfraz et al. 2018). Its current geographic distribution is not well-described but important to know since soft rot bacteria are easily transported long distances in latently infected seed tubers and can cause significant economic losses in potato production worldwide.

scholarly journals First Report of Pectobacterium brasiliense causing soft rot on mizuna (Brassica rapa var. japonica) in the United States

Plant Disease ◽  
2021 ◽  
Author(s):  
Diksha Klair ◽  
Joshua Silva ◽  
Eduardo Dario Arizala ◽  
Gamze Boluk ◽  
Shefali Dobhal ◽  
...  
Keyword(s):  
Brassica Rapa ◽  
Housekeeping Genes ◽  
Soft Rot ◽  
The United States ◽  
Bacterial Suspension ◽  
Original Strain ◽  
Distilled Water ◽  
Tetrazolium Chloride ◽  
First Report ◽  
Query Coverage

Mizuna (Brassica rapa var. japonica), a member of family Brassicaceae, is a leafy vegetable having phenolic and other compounds beneficial to human health, such as natural antioxidants (Khanam et al. 2012). In October 2020, a field of mizuna (variety: Early) on Oahu island was observed having 20-30% diseased plants. Four randomly selected infected mizuna plants, showing the symptoms of wilt and stem rot (Figure 1A-D), were collected and isolations were made to determine the pathogen. Small sections of infected stems were cut, surface sterilized with 0.6% sodium hypochlorite solution for 30 sec, followed by three consecutive rinses in distilled water. The tissues were macerated in a sterile 1.5 ml centrifuge tube containing 100 μl sterile water—macerated tissues were streaked onto crystal violet pectate medium (CVP) (Hélias et al. 2011) and incubated at 26 ± 2°C for 48 h. Isolated bacterial colonies that formed pits on the CVP plates were re-streaked onto dextrose peptone agar: Peptone (10 g/L), Dextrose (5 g/L) and Agar (17 g/L) (DPA–without tetrazolium chloride; Norman and Alvarez 1989) to obtain purified colonies for DNA isolation using DNeasy Blood and Tissue Kit (Qiagen, Germantown, MA). The two housekeeping genes (dnaA and gapA) were amplified and sequenced following the protocols used by Dobhal et al. (2020) and Boluk et al. (2020), for identity confirmation and phylogenetic analysis. Cleaned PCR products were sent to the GENEWIZ facility (Genewiz, La Jolla, CA) for sequencing of sense and antisense strands. The obtained sequences were aligned, manually edited, and consensus sequences were analyzed with BLASTn using the NCBI GenBank nucleotide and genome databases for identity confirmation. The BLASTn results demonstrated 100% query coverage of all four strains (PL248-PL251); and showed 100% identity of PL248 and PL249, and 99% identity of PL250 and PL251 with Pectobacterium brasiliense. All the sequences were submitted to the NCBI GenBank database under the following accession numbers: dnaA gene MW560271 - MW560274 (PL248 – PL251); and gapA gene MW560275 - MW560278 (PL248 - PL251). Pathogenicity was assessed by artificially inoculating 100 µl bacterial suspension of each strain (PL248 - 1.12x 10⁸ CFU/ml; PL249 - 1.32x 10⁸ CFU/ml; PL 250 - 1.2x 10⁸ CFU/ml and PL251 - 1.15x 10⁸ CFU/ml) onto four-week-old mizuna (variety: Leafy Asian Greens) plants in three replicates, using sterile pipette tips, which was stabbed into stem halfway and wrapped with parafilm. The inoculated plants were well maintained under controlled greenhouse conditions. As negative controls, three plants were inoculated with 100 µl distilled water. Soft rot and wilt symptoms (Figure 1E-H) were observed 24 hours post inoculation. No symptoms were observed on control plants (Figure 1F). All four strains were re-isolated from the inoculated plants and confirmed as P. brasiliense based on resequencing of the dnaA region and 100% homology with the sequences of original strain. In the phylogenetic tree (Figure 2), based on two housekeeping genes (dnaA and gapA), the bacterial strains from mizuna grouped with other P. brasiliense retrieved from the NCBI GenBank database. To our knowledge, this is the first report of P. brasiliense infecting mizuna plants in Hawaii or in the USA and is important because this species is one of the most aggressive pectolytic pathogens in the genus Pectobacterium. Understanding the diversity of different pectolytic phytopathogens is essential to formulating risk mitigation strategies as P. brasiliense could potentially pose a threat to additional vegetable crops, especially the crucifers vegetables (Arizala et al. 2019; Klair et al, 2021).

scholarly journals First Report of Pectobacterium carotovorum subsp. carotovorum on Spathiphyllum wallisii in Argentina

Plant Disease ◽  
2009 ◽  
Vol 93 (8) ◽  
pp. 842-842 ◽  
Author(s):  
A. M. Alippi ◽  
A. C. López
Keyword(s):  
16S Rrna ◽  
Natural Infection ◽  
Soft Rot ◽  
Unknown Etiology ◽  
Rrna Gene ◽  
Pectobacterium Carotovorum ◽  
Distilled Water ◽  
Strain Atcc ◽  
First Report ◽  
Carotovorum Subsp

Peace lily (Spathiphyllum wallisii Regel) is a popular ornamental potted plant in Argentina. During May of 2008 (austral autumn), necrotic lesions of unknown etiology were observed on S. wallisii in a nursery in Pontevedra (34°45′6″S, 58°42′42″W). Plants first showed water-soaked areas starting from the leaf tips. Infected tissue became irregular, brown, dark-to-black lesions on leaves ~12 to 14 mm in diameter surrounded by yellowish haloes. Disease incidence approached 30%. Abundant bacterial streaming was observed from lesions when examined at ×100. Bacteria isolated from lesions formed white-to-cream, glistening, convex colonies on yeast dextrose calcium carbonate agar. Three bacterial strains isolated from different symptomatic plants were selected for comparative analysis with Pectobacterium carotovorum subsp. carotovorum type strain ATCC 15713. All were facultatively, anaerobic, gram-negative rods, pectolytic on crystal violet pectate agar, nonfluorescent on King's medium B, and elicited a hypersensitive response in tobacco plants. All strains were oxidase and arginine dihydrolase negative, fermented glucose, did not hydrolyze starch, did not produce lecithinase, indole or the blue pigment indigoidine, reduced nitrates, hydrolyzed gelatin and esculin, able to rot onion slices, caused soft rot of potato tubers, resistant to erythromycin, and grew at 37°C. Acid was produced from cellobiose, d-glucose, d-melibiose, d-mannitol, d-mannose, l-rhamnose, d-sucrose, and l-arabinose but not from inositol and d-sorbitol. Bacteria utilized N-acetyl-glucosamine and citrate but not tartrate, benzoate, or propionate. Their identity was confirmed by 16S rRNA gene sequencing of strain F402Pcc (GenBank Accession No. FJ717337) showing a 99% homology with that of strain ATCC 3326 (FJ 5958691). Pathogenicity was verified on S. wallisii, Dieffenbachia picta, Aglaonema commutatum, and Anthurium andraeanum within the Araceae family by spraying two plants per strain tested with bacterial suspensions (108 CFU/ml) in sterile distilled water with and without wounding the leaves with sterile needles. Controls were sprayed with sterile distilled water. After 48 h in a humidity chamber, inoculated plants and controls were maintained at 25 ± 3°C in a greenhouse. Water-soaked areas developed from 24 to 48 h after inoculation and became necrotic within 4 to 5 days. Lesions expanded to resemble natural infection in S. wallisii within 20 days, while in the rest of the hosts tested, lesions were smaller and remained brown surrounded by yellowish haloes. All strains were reisolated from each host tested. The original and all reisolated strains were compared by enterobacterial repetitive intergeneric consensus-PCR (4) confirming that DNA fingerprints of the reisolated strains were identical to those of the original strains. No lesions were observed on controls. The pathogen was identified as P. carotovorum subsp. carotovorum based on biochemical, physiological, pathogenicity tests, and 16S rRNA sequencing (1–3).To our knowledge, this is the first report of this pathogen on S. wallisii in Argentina although it has been reported as causing tomato pith necrosis (1) and soft rot of vegetables after harvest (3). References: (1) A. M. Alippi et al. Plant Dis. 81:230, 1997. (2) L. Gardan et al. Int. J. Syst. Evol. Microbiol. 53:381, 2003. (3) L. Halperin and L. S. Spaini. Rev. Argent. Agron. 6:261, 1939. (4) F. J. Louws et al. Appl. Environ. Microbiol. 60:2286, 1994.

scholarly journals First Report of Bud Soft Rot on Agave angustifolia Caused by Pantoea dispersa in Mexico

Plant Disease ◽  
2021 ◽  
Author(s):  
FRANCISCO PALEMON ◽  
Santo Angel Ortega-Acosta ◽  
Santiago Dominguez-Monge ◽  
Alvaro Castañeda-Vildozola ◽  
Guadalupe Reyes-Garcia ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Soft Rot ◽  
Bacterial Suspension ◽  
Rrna Gene ◽  
Sterile Water ◽  
First Report ◽  
Agave Angustifolia ◽  
Pantoea Dispersa ◽  
Initial Symptoms

The agave (Agave spp.) is an important crop in México, with 120,897 ha grown mainly for alcoholic beverage production (SIAP, 2019). In September 2020, in the municipality of Huitzuco de los Figueroa (18.328692 N; 99.3998 W), Guerrero State, México, a serious disease was observed affecting Agave angustifolia. Disease incidence was 8% of 150 plants sampled over an approximate area of 2.5 ha. Initial symptoms of soft rot of the bud developed and produced an abundant exudate which appeared from the apical part to the base of the plant. In severe infections, the plants showed total maceration of the bud, and consequently death of the plants was observed. Symptomatic plant tissue was superficially disinfected with 1% NaOCl for 30 s, and rinsed in sterile water three times. The disinfected tissues were macerated and with a loop spread in Nutrient Agar. The plates were incubated at 28 ° C for 2 days. Yellowish bacterial colonies were isolated, and eight colonies were selected for characterization. The bacterial strains were gram negative and rod-shaped, negative for fluorescent pigment tests and Kovacs' oxidase. Two isolates designated AGA1 and AGA2 were identified by PCR amplification and sequencing of the partial 16S rRNA gene with the primer 27F / 1492R (Lane 1991), and partial fusA, rpoB, and gyrB genes (Delétoile et al. 2009). Sequences were deposited in GenBank, with the accession numbers for 16S rRNA, AGA1 as MW548406 and AGA2 as MW548407; for specific genes fusA (AGA1 = MW558445, AGA2 = MW558446), rpoB (AGA1 = MW558447, AGA2 = MW558448) and gyrB (AGA1 = MW558449, AGA2 = MW558450), and they were compared with the sequences available in GenBank using BLASTn. 16S rRNA gene sequences for AGA1 and AGA2 aligned with Pantoea dispersa (MT921704.1, 99.9% identity). Housekeeping genes also aligned 99 to 100% to P. dispersa (fusA = 100%, CP045216.1; rpoB = 99.8% MH015167.1 and gyrB = 99%, MK928270.1). Phylogenetic analysis of concatenated genes showed that strains AGA1 and AGA2 cluster with P. dispersa. To confirm pathogenicity, eight plants of six-month-old A. angustifolia were inoculated with strain AGA1 using sterile toothpicks dipped in 108 CFU/ml bacterial suspension. The toothpicks were inserted in the middle part of the bud. Four plants were inoculated with sterile water as control. The plants were covered with plastic bags and housed in a greenhouse (average temperature and relative humidity of 25 ° C and 85%, respectively). Pathogenicity tests were repeated two times. After seven days, all inoculated plants developed symptoms similar to those observed in the field. Control plants did not show symptoms. From the plants that showed symptoms, the pathogen was reisolated again and was identified by morphological and molecular characterization, following the method previously described, fulfilling Koch's postulates. In México, Erwinia cacticida and Pantoea ananatis has been previously reported on A. tequilana that as causing soft rot and red leaf ring, respectively (Jimenez-Hidalgo et al. 2004; Fucikovsky and Aranda 2006). To our knowledge, this is the first report of P. dispersa causing bud soft rot on A. angustifolia in México. More studies monitoring and control strategies of bud soft rot on A. angustifolia are required.

scholarly journals First Report of a Bacterial Soft Rot on Tolumnia Orchids Caused by a Dickeya sp. in the United States

Plant Disease ◽  
2009 ◽  
Vol 93 (12) ◽  
pp. 1354-1354 ◽  
Author(s):  
R. A. Cating ◽  
A. J. Palmateer ◽  
R. T. McMillan ◽  
E. R. Dickstein
Keyword(s):  
Pathogenic Bacteria ◽  
Soft Rot ◽  
The United States ◽  
South Florida ◽  
Nutrient Agar ◽  
Bacterial Suspension ◽  
Brown Pigment ◽  
Rrna Gene ◽  
University Of Florida ◽  
First Report

Tolumnia orchids are small epiphytic orchids grown for their attractive flowers. In the fall of 2008, approximately 100 Tolumnia orchids with soft, brown, macerated leaves were brought to the University of Florida Extension Plant Diagnostic Clinic in Homestead. Ten plants were randomly selected and bacteria were isolated from the margins of symptomatic tissues of each of the 10 plants on nutrient agar according to the method described by Schaad et al. (2). Four reference strains were used in all tests, including the molecular tests: Erwinia carotovora subsp. carotovora (obtained from J. Bartz, Department of Plant Pathology, University of Florida, Gainesville), E. chrysanthemi (ATCC No. 11662), Pectobacterium cypripedii (ATCC No. 29267), and Acidovorax avenae subsp. cattleyae (ATCC No. 10200). All 10 of the isolated bacteria were gram negative, grew at 37°C, degraded pectate in CVP (crystal violet pectate) medium, grew anaerobically, produced brown pigment on NGM (nutrient agar-glycerol-manganese chloride) medium (1), were sensitive to erythromycin, and produced phosphatase. Three of the strains were submitted for MIDI analysis (Sherlock version TSBA 4.10; Microbial Identification, Newark DE) (SIM 0.732 to 0.963), which identified them as E. chrysanthemi. A PCR assay was performed on the 16S rRNA gene with primers 27f and 1495r described by Weisburg et al. (3) from two of the isolates and a subsequent GenBank search showed 99% identity of the 1,508-bp sequence to that of Dickeya chrysanthemi (Accession No. FM946179) (formerly E. chrysanthemi). The sequences were deposited in GenBank (Accession Nos. GQ293897 and GQ293898). Pathogenicity was confirmed by injecting approximately 100 μl of a bacterial suspension at 1 × 108 CFU/ml into leaves of 10 Tolumnia orchid mericlones. Ten plants were also inoculated with water as controls. Plants were placed in a greenhouse at 29°C with 60 to 80% relative humidity. Within 24 h, soft rot symptoms appeared on all inoculated leaves. The water controls appeared normal. A Dickeya sp. was reisolated and identified using the above methods (biochemical tests and MIDI), fulfilling Koch's postulates. To our knowledge, this is the first report of a soft rot caused by a Dickeya sp. on Tolumnia orchids. Although 16S similarity and MIDI results suggest the isolated bacteria are D. chrysanthemi because of its close similarity with other Dickeya spp., these results are not conclusive. Further work should be conducted to confirm the identity of these isolates. Through correspondence with South Florida Tolumnia growers, it appears this disease has been a recurring problem, sometimes affecting international orchid shipments where plant losses have been in excess of 70%. References: (1) Y. A. Lee and C. P. Yu. J. Microbiol. Methods 64:200, 2006. (2) N. W. Schaad et al. Erwinia soft rot group. Page 56 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. American Phytopathological Society. St. Paul, MN, 2001. (3) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

scholarly journals First Report of Pantoea dispersa Causing Brown Blotch Disease in Flammulina filiformis in China

Plant Disease ◽  
2021 ◽  
Author(s):  
Haijing Hu ◽  
Miao Xu ◽  
Haiyan Song ◽  
Zhijun Zhai ◽  
Minghui Chen ◽  
...  
Keyword(s):  
Relative Humidity ◽  
Yield Reduction ◽  
Bacterial Suspension ◽  
Rrna Gene ◽  
Distilled Water ◽  
Fruiting Bodies ◽  
Culture Bottle ◽  
First Report ◽  
Brown Blotch ◽  
Brown Blotch Disease

Flammulina filiformis (previously known as F. velutipes) is one of the most frequently cultivated and consumed edible mushrooms in China. In October 2020, brown blotch disease was observed on the pileus of F. filiformis at a mushroom factory in Ganzhou (25.74°N; 114.95°E), Jiangxi, China, with a disease incidence of approximately 6%. Symptoms initially appeared as small, irregular spots on the infected pileus, with color ranging from pale yellow to light brown. Such spots were enlarged and pitted at high relative humidity within several days, and finally caused malformation of the caps and yield reduction. To isolate the causal agent, the blotches on F. filiformis caps were homogenized and diluted with sterilized distilled water, and the resulting suspension (100 μl) was spread onto LB agar plates. After incubation at 28°C for 48 h, three colonial types were obtained: (i) yellow, convex, and smooth colonies, (ii) light yellowish, irregular, and rough colonies, and (iii) milky white, glistening, and smooth colonies. The first colonial type was predominant. A single colony of each type was randomly selected and streaked on fresh LB agar plates to obtain pure cultures namely PF1, PF2 and PF3 respectively. To test pathogenicity of the three isolates, young F. filiformis fruiting bodies (8- or 10-day-old primordium) grown in culture bottles were inoculated by spraying a bacterial suspension (108 CFU/ml, 3 ml per bottle on average), and cultivated in a mushroom house at 15±2°C and 95% relative humidity. The fruiting bodies sprayed with sterilized distilled water served as controls. The pathogenicity tests repeated three times, and at least five culture bottles were included in each experiment. Among the three types of bacteria, only strain PF1 induced symptoms similar to the original disease. The brown spots were observed on treated pileus 10 days after inoculation, and a fresh weight reduction of 30.9% per culture bottle was observed. In contrast, those fruiting bodies treated with water remained asymptomatic. Same yellow colonies were also re-isolated from the infected pileus, and identified by subsequent methods. The strain PF1 was gram negative, motile, and short rods. Biochemical analysis showed that the strains belonged to genus Pantoea (positive for citric acid, inositol, mannitol, methyl red test, and Voges-Proskauer test but negative for lysine, ornithine, phenylalanine, H2S, urease, D-melibiose, sorbitol, adonitol, and raffinose). Further PCR amplification and sequencing of four genes, 16S rRNA gene with primer 27F/1492R, fusA gene with primer fusA3/fusA4, gyrB gene with primer gyrBf1/gyrBr1, and rpoB genes with primer Vic3/Vic2 (Delétoile et al. 2009; Palemon et al. 2021), were performed to identify the species. A BLASTn showed 100%, 100%, 99.51%, and 99.26% homology, respectively, with those of P. dispersa (MT072166, CP045216, CP076369, MH015168). The four gene sequences were deposited in GenBank (accession numbers: MZ373179, MZ393661, MZ393662, MZ393663). A phylogenetic analysis based on the four concatenated genes also showed that the strain PF1 well clustered with the type strain of P. dispersa. This species has been reported to cause leaf blight in rice (Toh et al. 2019), soft rot in Agave angustifolia (Palemon et al. 2021), and bulb decay in onion (Chang et al. 2018). To the best of our knowledge, this is the first report of P. dispersa causing brown blotch diseases on cultivated F. filiformis, which was previously known to be caused by Pseudomonas tolaasii (Lee et al. 2002). Our results also indicate P. dispersa could induce malformation of pileus and lead to a severe yield loss if not controlled effectively. Therefore, it should be considered in future disease management of F. filiformis cultivation.

scholarly journals First Report of Pepper Fruit Rot Caused by Fusarium concentricum in China

Plant Disease ◽  
2013 ◽  
Vol 97 (12) ◽  
pp. 1657-1657 ◽  
Author(s):  
J. H. Wang ◽  
Z. H. Feng ◽  
Z. Han ◽  
S. Q. Song ◽  
S. H. Lin ◽  
...  
Keyword(s):  
Fusarium Species ◽  
Fruit Rot ◽  
Post Inoculation ◽  
Distilled Water ◽  
Conidial Suspension ◽  
Average Growth ◽  
First Report ◽  
Control Fruit ◽  
Pepper Fruit ◽  
Fruit Samples

Pepper (Capsicum annuum L.) is an important vegetable crop worldwide. Some Fusarium species can cause pepper fruit rot, leading to significant yield losses of pepper production and, for some Fusarium species, potential risk of mycotoxin contamination. A total of 106 diseased pepper fruit samples were collected from various pepper cultivars from seven provinces (Gansu, Hainan, Heilongjiang, Hunan, Shandong, Shanghai, and Zhejiang) in China during the 2012 growing season, where pepper production occurs on approximately 25,000 ha. Pepper fruit rot symptom incidence ranged from 5 to 20% in individual fields. Symptomatic fruit tissue was surface-sterilized in 0.1% HgCl2 for 1 min, dipped in 70% ethanol for 30 s, then rinsed in sterilized distilled water three times, dried, and plated in 90 mm diameter petri dishes containing potato dextrose agar (PDA). After incubation for 5 days at 28°C in the dark, putative Fusarium colonies were purified by single-sporing. Forty-three Fusarium strains were isolated and identified to species as described previously (1,2). Morphological characteristics of one strain were identical to those of F. concentricum. Aerial mycelium was reddish-white with an average growth rate of 4.2 to 4.3 mm/day at 25°C in the dark on PDA. Pigments in the agar were formed in alternating red and orange concentric rings. Microconidia were 0- to 1-septate, mostly 0-septate, and oval, obovoid to allantoid. Macroconidia were relatively slender with no significant curvature, 3- to 5-septate, with a beaked apical cell and a foot-shaped basal cell. To confirm the species identity, the partial TEF gene sequence (646 bp) was amplified and sequenced (GenBank Accession No. KC816735). A BLASTn search with TEF gene sequences in NCBI and the Fusarium ID databases revealed 99.7 and 100% sequence identity, respectively, to known TEF sequences of F. concentricum. Thus, both morphological and molecular criteria supported identification of the strain as F. concentricum. This strain was deposited as Accession MUCL 54697 (http://bccm.belspo.be/about/mucl.php). Pathogenicity of the strain was confirmed by inoculating 10 wounded, mature pepper fruits that had been harvested 70 days after planting the cultivar Zhongjiao-5 with a conidial suspension (1 × 106 spores/ml), as described previously (3). A control treatment consisted of inoculating 10 pepper fruits of the same cultivar with sterilized distilled water. The fruit were incubated at 25°C in a moist chamber, and the experiment was repeated independently in triplicate. Initially, green to dark brown lesions were observed on the outer surface of inoculated fruit. Typical soft-rot symptoms and lesions were observed on the inner wall when the fruit were cut open 10 days post-inoculation. Some infected seeds in the fruits were grayish-black and covered by mycelium, similar to the original fruit symptoms observed at the sampling sites. The control fruit remained healthy after 10 days of incubation. The same fungus was isolated from the inoculated infected fruit using the method described above, but no fungal growth was observed from the control fruit. To our knowledge, this is the first report of F. concentricum causing a pepper fruit rot. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (2) K. O'Donnell et al. Proc. Nat. Acad. Sci. USA 95:2044, 1998. (3) Y. Yang et al. 2011. Int. J. Food Microbiol. 151:150, 2011.

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