First Report of a Soft Rot of Phalaenopsis aphrodita Caused by Dickeya dieffenbachiae in China

Phalaenopsis orchids, originally from tropical Asia, are mainly planted in Thailand, Singapore, Malaysia, the Philippines, and Taiwan and have gained popularity from consumers all over the world. The cultivation area of Phalaenopsis orchids has been rising and large-scale bases have been established in mainland China, especially South China because of suitable environmental conditions. In September 2011, a soft rot of Phalaenopsis aphrodita was found in a Phalaenopsis planting base in Guangzhou with an incidence of ~15%. Infected plants initially showed water-soaked, pale-to-dark brown pinpoint spots on leaves that were sometimes surrounded by a yellow halo. Spots expanded rapidly with rising humidity and temperatures, and in a few days, severely extended over the blade with a light tan color and darker brown border. Lesions decayed with odorous fumes and tissues collapsed with inclusions exuding. The bacterium advanced to the stem and pedicle. Finally, leaves became papery dry and the pedicles lodged. Six diseased samples were collected, and bacteria were isolated from the edge of symptomatic tissues after sterilization in 0.3% NaOCl for 10 min, rinsing in sterile water three times, and placing on nutrient agar for culture. Twelve representative isolates were selected for further characterization. All strains were gram negative, grew at 37°C, were positive for indole production, and utilized malonate, glucose, and sucrose but not glucopyranoside, trehalose, or palatinose. Biolog identification (version 4.20.05, Hayward, CA) was performed and Pectobacterium chrysanthemi (SIM 0.868) was confirmed for the tested isolates (transfer to genus Dickeya). PCR was used to amplify the 16S rDNAgene with primers 27f and 1492r, dnaX gene with primers dnaXf and dnaXr (3), and gyrB gene with primers gyrBf (5′-GAAGGYAAAVTKCATCGTCAGG-3′) and gyrB-r1 (5′-TCARATATCRATATTCGCYGCTTTC-3′) designed on the basis of the published gyrB gene sequences of genus Dickeya. BLASTn was performed online, and phylogeny trees (100% bootstrap values) were created by means of MEGA 5.05 for these gene sequences, respectively. Results commonly showed that the representative tested strain, PA1, was most homologous to Dickeya dieffenbachiae with 98% identity for 16S rDNA(JN940859), 97% for dnaX (JN989971), and 96% for gyrB (JN971031). Thus, we recommend calling this isolate D. dieffenbachiae PA1. Pathogenicity tests were conducted by injecting 10 P. aphrodita seedlings with 100 μl of the bacterial suspension (1 × 108 CFU/ml) and another 10 were injected with 100 μl of sterile water as controls. Plants were inoculated in a greenhouse at 28 to 32°C and 90% relative humidity. Soft rot symptoms were observed after 2 days on the inoculated plants, but not on the control ones. The bacterium was isolated from the lesions and demonstrated identity to the inoculated plant by the 16S rDNA sequence comparison. Previously, similar diseases of P. amabilis were reported in Tangshan, Jiangsu, Zhejiang, and Wuhan and causal agents were identified as Erwinia spp. (2), Pseudomonas grimontii (1), E. chrysanthemi, and E. carotovora subsp. carovora (4). To our knowledge, this is the first report of D. dieffenbachiae causing soft rot disease on P. aphrodita in China. References: (1) X. L. Chu and B. Yang. Acta Phytopathol. Sin. 40:90, 2010. (2) Y. M. Li et al. J. Beijing Agric. Coll. 19:41, 2004. (3) M. Sławiak et al. Eur. J. Plant Pathol. 125:245, 2009. (4) Z. Y. Wu et al. J. Zhejiang For. Coll. 27:635, 2010.

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First Report of a Soft Rot of Philodendron ‘Con-go’ in China Caused by Dickeya dieffenbachiae

Plant Disease ◽

10.1094/pdis-09-11-0795 ◽

2012 ◽

Vol 96 (3) ◽

pp. 452-452 ◽

Cited By ~ 5

Author(s):

B. R. Lin ◽

H. F. Shen ◽

J. N. Zhou ◽

X. M. Pu ◽

Z. N. Chen ◽

...

Keyword(s):

16S Rdna ◽

Phylogenetic Trees ◽

Soft Rot ◽

Gyrb Gene ◽

Control Treatment ◽

Gene Sequences ◽

16S Rdna Gene ◽

First Report ◽

Rdna Gene ◽

Plastic Bags

Philodendron is a popular foliage plant cultivated in interiorscapes of homes, offices, and malls throughout China. A severe outbreak of a soft rot of Philodendron ‘Con-go’ occurred in Guangzhou, China from 2010 to 2011. The disease was characterized by leaf infections starting as pinpoint spots that are water soaked and yellow to pale brown. The lesions are sometimes surrounded by a diffuse yellow halo. When the humidity is high and temperatures are warm to hot, the spots expand rapidly, becoming slimy, irregular, and sunken with light tan centers, darker brown borders, and diffused yellow margins and may involve the entire leaf in a few days. An invasion of the midrib and larger veins by the causal bacterium often results in advancement into the petiole and stem. A survey of three areas of production of Philodendron ‘Con-go’ (5 ha) in Guangzhou revealed that 91% of the fields were affected at an incidence ranging from 15 to 30%. Of 41 bacterial isolates obtained from lesions, three were selected randomly for further characterization. All strains were gram negative, negative for oxidase and positive for catalase and tryptophanase (indole production), and utilized citrate, tartrate, malonate, glucose, sucrose, fructose, and maltose but not glucopyranoside, trehalose, or palatinose. Biolog analysis (version 4.20.05, Hayward, CA) identified the isolates as Pectobacterium chrysanthemi (SIM 0.804 to 0.914). According to Samson et al. (1), it was renamed as a Dickeya sp. PCR was performed on the 16S rDNA gene with primers 27f and 1495r (3) and 1,423 bp of the 16S rDNA gene (GenBank No. JN709491) showed 99% identity to P. chrysanthemi (GenBank No. AF373202), and 98% to Dickeya dieffenbachiae (GenBank No. JF311644). Additionally, the gyrB gene was amplified with primers gyrB-f1 (5′-atgtcgaattcttatgactcctc-3′) and gyrB-r1 (5′-tcaratatcratattcgcygctttc-3′) designed based on all the submitted gyrB gene sequences of Dickeya spp. The dnaX gene was amplified with primers dnaXf and dnaXr (2). The products were sequenced and phylogeny analyses were performed by means of MEGA 5.05. Results showed that the gyrB and the dnaX genes of the strains were 98% homologous to those of D. dieffenbachiae (GenBank Nos. JF311652 and GQ904757). Therefore, on the basis of phylogenetic trees of the 16S rDNA, gyrB, and dnaX gene sequences, the bacterial isolate named PC1 is related to D. dieffenbachiae (100% bootstrap values). Pathogenicity of each of the three strains on Philodendron ‘Con-go’ was confirmed by injecting 60 50-day-old seedlings each with 0.1 ml of the isolate suspension (108 CFU/ml) into the leaves. Another 60 were injected with sterile water to serve as the control treatment. Plants were enclosed in plastic bags and returned to the greenhouse under 50% shade at 32°C day and 28°C night temperatures with high humidity. After 72 h, all the injected plants started to show symptoms similar to those observed on field plants, but no symptoms appeared on the control plants. The reisolates were identical to the inoculated strains in biochemical characteristics. Bacteria characteristic of the inoculated strains were not reisolated from the control plants. To our knowledge, this is the first report of D. dieffenbachiae causing soft rot of Philodendron ‘Con-go' in China. References: (1) R. Samson et al. Evol. Microbiol. 55:1415, 2005. (2) M. Sławiak et al. Eur. J. Plant Pathol. 125:245, 2009. (3) W. G. Weisbury et al. J. Bacteriol. 173:697, 1991.

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First Report of Dickeya fangzhongdai Causing Peduncle Soft Rot of Banana in China

Plant Disease ◽

10.1094/pdis-11-21-2513-pdn ◽

2021 ◽

Author(s):

Di Yang ◽

Chan Juan Du ◽

Yunfeng Ye ◽

Lian Fu Pan ◽

Jin Zhang ◽

...

Keyword(s):

16S Rdna ◽

Dna Sequences ◽

Southern China ◽

Intergenic Spacer ◽

Soft Rot ◽

Bacterial Suspension ◽

Sterile Water ◽

Internal Tissue ◽

Flower Bud ◽

First Report

Banana (Musa spp.) is a popular fruit all over the world, and it’s also an important cash crop with a planting area of 358,924 ha in southern China. In July 2020, a peduncle soft rot disease occurred on dwarf banana (Musa sp. cv. Guangfen) in Guigang city (N22°50'29″, E109° 43'34″), Guangxi province, China. More than 20% plants were infected in the banana plantation. The first external sign of the disease appeared on the incisional wound after the flower bud was cut off from the peduncle. The symptom initially appeared as a black lesion on the wound, then extended into the internal tissue of the whole peduncle. In the later stages, the internal tissue became soft and rot, occasionally formed a necrotic cavity, and eventually led to the black rot of the whole peduncle with a foul smell. To isolate the pathogen, the internal lesion tissues of 5 mm × 5 mm were collected between the border of symptomatic and healthy tissue, treated with 75% ethanol for 10 s, and 0.1% HgCl2 for 3 min, then rinsed with sterile water for three times. Sterilized tissue fragments were cut to pieces with sterilized surgical shears and soaked in 5 mL sterile water, then shaken for 10 min in a vortex oscillator. The suspension was diluted 1000 times with sterilized water，then plated on nutrient-agar medium and incubated at 28℃ in darkness for 24 h. Among the 32 isolates, 23 pure bacterial cultures with similar morphology were predominantly obtained from the samples. These bacteria were gram-negative, and their colonies were initially yellowish white with irregular edges and smooth surfaces, then turned to grayish blue after 72 h incubated at 28℃. The representative isolates GZF2-2 and GZF1-8 were selected for further identification. Genomic DNA was isolated from the bacteria and the 16S rDNA was amplified with primers 27F/1492R (Weisburg et al. 1991) and sequenced. The obtained sequences (GenBank Accession No. MZ768922 and OK668082) showed >99% identities to several records of Dickeya fangzhongdai deposited in NCBI GenBank (1400/1404 bps for GZF2-2 to KT992690, 1409/1417 bps for GZF1-8 to MT613398) based on BLAST analysis. In addition, the recA, fusA, gapA, purA, rplB, dnaX genes and the 16S-23S intergenic spacer (IGS) regions of the two isolates were also amplified and sequenced (GenBank Accession Nos. OK634381-OK634382, OK634369- OK634370, OK634373-OK634374, OK634377-OK634378, OK634385-OK634386, OK634365- OK634366 and OK631722-OK631723) as described by Tian et al. (2016). All the DNA sequences matched that of D. fangzhongdai strains JS5T (percent identities>99.06%), PA1 and ECM-1 in GenBank. Neighbor-joining phylogenetic analysis by software MegaX (Kumar et al. 2018) based on the 16S rDNA sequences revealed that the two isolates were in the same clade with reported D. fangzhongdai strains. Multilocus sequence analysis of the other seven regions also showed the two representative isolates were belong to D. fangzhongdai. Therefore, the isolates were identified as D. fangzhongdai. Pathogenicity of isolate GZF2-2 was investigated to demonstrate Koch’s postulate. The end of the banana peduncles of 6 healthy plants were cut off, and 10 mL bacterial suspension (108 CFU/mL) was inoculated to the fresh wound on the plants using sterile brushes. Six control plants were inoculated with sterilized water. All the inoculated peduncles were covered with plastic bags to maintain high humidity. After 28 days, all the peduncles inoculated with strain GZF2-2 showed soft rot symptoms similar to those observed in the field, while the controls remained symptomless. The same bacteria were re-isolated from the symptomatic peduncles and confirmed by sequencing the 16S rDNA. D. fangzhongdai has been reported to cause soft rot on onion (Ma et al. 2020) and bleeding cankers on pear trees (Chen et al. 2020). To the best of our knowledge, this is the first report of D. fangzhongdai causing peduncle soft rot on banana in China.

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First Report of Bacterial Foot Rot of Rice Caused by a Dickeya zeae in China

Plant Disease ◽

10.1094/pdis-03-12-0315-pdn ◽

2012 ◽

Vol 96 (12) ◽

pp. 1818-1818 ◽

Cited By ~ 10

Author(s):

X. M. Pu ◽

J. N. Zhou ◽

B. R. Lin ◽

H. F. Shen

Keyword(s):

16S Rdna ◽

Guangdong Province ◽

Gyrb Gene ◽

Bacterial Suspension ◽

Bacterial Disease ◽

Sterile Water ◽

Foot Rot ◽

Rice Seedlings ◽

First Report ◽

Negative Controls

A bacterial disease of rice, bacterial foot rot, was found in Guangdong Province, China in September 2011, with an incidence about 10%. The typical symptom was a dark brown decay of the tillers. In the early stages of the disease, a brown sheath rot seemed to spread from the ligulae regions. The lesions quickly extended down to the nodes, culms, and finally to the crowns. Neighboring tillers of the same crown were invaded systemically, causing foot rot symptoms. A soft rot with an unpleasant odor developed in young tissues of infected tillers. In the advanced stage, many tillers decayed, so that entire diseased plants could easily be pulled from the soil. Six diseased samples were collected and bacteria were isolated from the edge of symptomatic tissues, after samples were sterilized in 0.3% NaOCl for 10 min, rinsed in sterile water three times, and placed on nutrient agar (beef extract 3 g, yeast extract 1 g, peptone 5 g, glucose 10 g, agar 16 g, distilled water 1 L, pH 6.8 to 7.0). For identification, a total of 12 representative isolates were selected. All strains were Gram negative, grew at 37°C, were positive for indole production, and utilized malonate, glucose, and sucrose, but not glucopyranoside, trehalose, or palatinose. Biolog identification (Version 4.20.05, Hayward, CA) identified isolate EC1 as Pectobacterium chrysanthemi (SIM 0.827), which has since been transferred to genus Dickeya. PCR was used to amplify the 16S rDNA gene with primers 27f and 1492r, the dnaX gene with primers dnaXf and dnaXr (2), and the gyrB gene with primers gyrBf1 (5′-ATGTCGAATTCTTATGACTCCTC-3′) and gyrB-r1 (5′-TCARATATCRATATTCGCYGCTTTC-3′), which were designed based on published gyrB gene sequences of genus Dickeya. A BLASTn search of all three loci [16S rDNA (JQ284040), dnaX (JQ284041), and gyrB (JQ284042)] revealed that EC1 had 100% sequence identify to Dickeya zeae [16S rDNA (AB713560), dnaX (AB713593), gyrB (AB713635)]. Pathogenicity tests were conducted by injecting 10 rice seedlings with 100 μl of the bacterial suspension (1 × 108 CFU/ml) in the stem base, and an additional 10 rice seedlings were injected with 100 μl of sterile water as negative controls. Inoculations were carried out in a greenhouse at 28 to 32°C and 90% relative humidity. Foot rot symptoms identical to those described above were observed after 7 days on inoculated plants, but not on the negative controls. The bacterium was reisolated from the lesions and had 100% sequence identity for all three loci to EC1. Previously, similar symptoms were reported on rice in Guangdong province of China, and the causal agent was identified as Erwinia chrysanthemi (1). To our knowledge, this is the first report of D. zeae causing foot rot disease on rice in China. References: (1) Q. G. Liu et al. J. South China Agric. Univ. 18:128, 1997. (2) M. Sławiak et al. Eur. J. Plant Pathol. 125:245, 2009.

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First Report of Bacterial Blight of Guar Caused by Xanthomonas axonopodis pv. cyamopsidis in China

Plant Disease ◽

10.1094/pdis-09-13-0983-pdn ◽

2014 ◽

Vol 98 (6) ◽

pp. 840-840 ◽

Cited By ~ 2

Author(s):

Y. Z. Ren ◽

Y. L. Yue ◽

G. X. Jin ◽

Q. Du

Keyword(s):

16S Rdna ◽

Bacterial Blight ◽

Negative Control ◽

Bacterial Suspension ◽

Cyamopsis Tetragonoloba ◽

Sterile Water ◽

Bacterial Strains ◽

Xanthomonas Axonopodis ◽

First Report ◽

Pathogenicity Tests

Bacterial blight was observed on field-grown guar (Cyamopsis tetragonoloba L.) for the first time in China. The disease outbreak occurred in the Xinjiang Uyghur Autonomous Region after several weeks of unusually heavy rains during late summer 2013. The disease incidence was generally 40 to 50%, although values as high as 80% were observed in several fields. Initial field symptoms included water-soaked spots on leaves, pods, petioles, and stems. During later stages of infection, the color of the spots became dark. We also observed large, angular, necrotic lesions at leaf tips, black streaks on petioles and stems, split stems, defoliation, wilting or top withering, vascular necrosis, and dieback. Samples of diseased leaves, stems, petioles, pods, and seeds were surface sterilized, ground, and then plated onto King's B medium. Plates were incubated at 28°C for 72 h. Fifteen bacterial strains with yellow-pigmented, opaque, and round colonies were isolated. These strains were aerobic, gram-negative rods with a single, polar flagellum. They were positive for H2S, esculin, oxidase, tobacco hypersensitivity, indole production from tryptophan, nitrate reduction to nitrite, and the utilization of glucose, mannose, trehalose, galactose, and starch. The maximum salt tolerance of the strains was 2 to 3%. Pathogenicity tests using eight strains were conducted in July 2013. A bacterial culture was suspended in sterile water with a final concentration of 108 CFU/ml. Eight 4-week-old guar plants were inoculated by (i) spraying the bacterial suspension on the leaves until runoff, or (ii) puncturing the stems with a needle that had been dipped into the bacterial suspension. Sterile water was used as a negative control. Plants were kept in a mist room with 100% relative humidity for 24 h. Stem and leaf symptoms similar to those of the original plants were observed on the inoculated guar plants within 10 days of inoculation. No symptoms developed on the negative control plants. Yellow bacterial colonies re-isolated from inoculated plant tissues were morphologically identical to the original. 16S rDNA was amplified using universal primers (Pa 5′-AGTTTGATCCTGGCTCAG-3′ and Ph 5′-TACCTTGTTACGACTTCGTCCCA-3′) and sequenced. A BLAST search of the NCBI GenBank database indicated that the 16S rDNA sequences of three strains (accession nos. KF563926, KF563927, and KF563928) had 99.9% identity to Xanthomonas axonopodis strain XV938 (AF123091). Under greenhouse conditions, bacterial strains wilted asparagus bean and pea but rarely infected bean, kidney bean, faba bean, mung bean, soybean, red bean, pea, garbanzo bean, and peanut. Based on morphology, pathogenicity tests, 16S rDNA sequencing, and host plant specificity, the pathogen was confirmed as X. axonopodis pv. cyamopsidis (synonym: X. campestris pv. cyamopsidis [Patel et al., 1953]). To our knowledge, this is the first report of bacterial blight of guar caused by X. axonopodis pv. cyamopsidis in China. Guar has recently been introduced in Xinjiang Province. Our findings indicate that bacterial blight may pose a threat to the economic sustainability of guar production in the region. References: (1) I. A. Milyutina et a1. FEMS Microbiol. Lett. 239:17, 2004. (2) I. M. G. Almeida et al. Summa Phytopathol. 18:255, 1992. (3) J. D. Mihail et al. Plant Dis. 69:811, 1985.

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First Report of Pantoea anthophila Causing Soft Rot Disease in Clausena lansium (Wampee) in China

Plant Disease ◽

10.1094/pdis-10-14-1025-pdn ◽

2015 ◽

Vol 99 (3) ◽

pp. 416-416 ◽

Cited By ~ 1

Author(s):

J. N. Zhou ◽

S. Y. Liu ◽

Y. F. Chen ◽

L. S. Liao

Keyword(s):

Disease Incidence ◽

Soft Rot ◽

The Philippines ◽

Luria Bertani ◽

Bacterial Suspension ◽

16S Rrna Sequence ◽

First Report ◽

Surface Sterilization ◽

Soft Rot Disease ◽

Rot Disease

Clausena lansium, also known as wampee (Clausena wampi), is a plant species native to China, Vietnam, the Philippines, Malaysia, and Indonesia, where it is widely cultivated, and also grown in India, Sri Lanka, Queensland, Florida, and Hawaii, but less frequently (3). The fruit can be consumed fresh or made into juice, jam, or succade. In summer to fall 2014, a soft rot disease was found in a wampee planting region in Yunan County, Guangdong Province, China. On Sept. 18, we collected diseased samples from a wampee orchard with about 20% disease incidence. The infected fruit initially showed pinpoint spots on the peel, water-soaked lesions, and light to dark brown discoloration. Spots expanded in 2 days, and tissues collapsed after 5 days. Severely affected fruit showed cracking or nonodorous decay. Five diseased samples were collected, and causal agents were isolated from symptomatic tissues 1 cm under the peel after surface sterilization in 0.3% NaOCl for 10 min and rinsing in sterile water three times. Tissues were placed on a Luria Bertani (LB) plate for culture. Ten representative isolates were selected for further characterization. No colony was isolated from healthy tissues. Colonies were round, smooth, with irregular edges, and produced a yellow pigment in culture. Biolog identification (Version 4.20.05) showed that all strains were gram negative, negative for indole production, and utilized glucose, maltose, trehalose, sucrose, D-lactose, and pectin but not sorbitol or gelatin. The isolates were identified as Pantoea agglomerans (SIM 0.69). Multilocus sequence analysis (MLSA) was conducted for rapid classification of the strains. Sequences of atpD, gyrB, infB, and rpoB were amplified using corresponding primers (2). All sequences of the 10 isolates were identical in each gene. BLASTn was performed, and maximum likelihood trees based on the concatenated nucleotide sequences of the four genes were constructed using MEGA6. Bootstrap values after 1,000 replicates were expressed as percentages. Results showed that the tested strain named CL1 was most homologous to P. anthophila, with 98% identity for atpD (KM521543), 100% for gyrB (KM521544), infB (KM521545), and rpoB (KM521546). The 16S rRNA sequence (KM521542) amplified by primers 27f and 1492r shared 99% identity with that of P. anthophila M19_2C (JN644500). P. anthophila was previously reclassified from P. agglomerans (3); therefore, we suggest naming this wampee pathogen P. anthophila. Subsequently, 10 wampee fruits were injected with 20 μl of bacterial suspension (1 × 108 CFU/ml) of strains CL1 and CL2, respectively, and another 10 were injected with 20 μl of LB medium as controls, all kept at 28°C for 4 days. Symptoms similar to those of natural infections were observed on inoculated fruits but not on the negative controls. Bacteria were isolated from diseased tissues and further identified as P. anthophila by gyrB sequencing. P. anthophila was reported to naturally infect balsam and marigold (1,2). To our knowledge, this is the first report of P. anthophila naturally causing soft rot disease and cracking on C. lansium (wampee). References: (1) C. Brady et al. Syst. Appl. Microbiol. 31:447, 2008. (2) C. Brady et al. Int. J. Syst. Evol. Microbiol. 59:2339, 2009. (3) J. Morton. Fruits of Warm Climates. Echo Point Books & Media, Miami, FL, 1987.

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Occurrence of Dickeya fangzhongdai Causing Soft Rot of Banxia (Pinellia ternata) in China

Plant Disease ◽

10.1094/pdis-01-21-0030-pdn ◽

2021 ◽

Author(s):

Fanfan Wang ◽

Tao Tang ◽

ting Mao ◽

Jie Guo ◽

XiaoLiang Guo ◽

...

Keyword(s):

16S Rdna ◽

Soft Rot ◽

Negative Control ◽

Reca Gene ◽

Bacterial Suspension ◽

Herbaceous Plant ◽

Pathogenicity Test ◽

Sterile Water ◽

Pinellia Ternata ◽

Traditional Chinese Herbal Medicine

Banxia [Pinellia ternata (Thunb.) Breit., Araceae] is a perennial herbaceous plant, where the tuber is commonly used in traditional Chinese herbal medicine. In the summer of 2020, an outbreak of soft rot of Banxia was observed in Zhugentan Town (30°50′N, 112°91′E), Qianjiang City, Hubei Province, with about 56% percentage of infected plants. Symptomatic plants initially appeared with small water-soaked spots on leaves that progressed into extensive translucent spots when facing a light source. The bacteria further spread to the stems and tubers. Infected tubers appeared normal, but inner macerated inclusions exuded when touched. The whole plant was macerated and collapsed within a few days. Ten leaves with typical symptoms were obtained from a diseased field, by surface sterilizing in 75% ethanol for 30 s and 0.3% NaClO for 5 min, washing the tissue sections three times in sterile water. Small pieces of tissue (5 × 5 mm) were removed from lesion borders, plated on nutrient ager medium, and cultivated at 37 ℃ for 48 h. Five representative isolates were selected for further identification. Colonies were all smooth and transparent. In addition, these strains were Gram-negative, and had the ability to reduce D-arabinose, melibiose, galactose, raffinose, rhamnose, inositol, and mannitol, but not reduce 5-keto-D-gluconate, L-xylose, amygdalin, and sorbitol. Genomic DNA was extracted from isolate stain ZG5. The 16S rDNA gene, recombinase A (recA) gene, and DNA polymerase III subunits gamma and tau (dnaX) were amplified by PCR with the primers 27f/1492r (Weisburg et al. 1991), recF/recR (Waleron et al. 2002), and dnaXf/dnaXr (Sławiak et al. 2009), respectively. The PCR products were sequenced, then submitted to GenBank (GenBank MW332472, MW349833, MW349834, respectively). BLAST search showed that the sequences of 16S rDNA, recA, and dnaX respectively matched ≥99% with D. fangzhongdai strains DSM 101947 (CP025003), QZH3 (CP031507), and PA1 (CP020872). Pathogenicity tests were performed on 10 healthy, 3-month-old P. ternate plants. Five plants were injected with 20 μl of bacterial suspension (108 CFU/ml) of isolate ZG5, and other plants were injected with sterile water as a negative control. All tested plants were incubated at 28 ℃ and individually covered with a plastic bag. After 24 h, soft rot symptoms all appeared on the pathogen-inoculated leaves, whereas no symptoms on the control leaves. The pathogenicity test was repeated three times and obtained same results. Koch’s postulates were fulfilled by reisolating D. fangzhongdai from inoculated plants. Meanwhile, PCR were performed on the reisolated bacteria as above described, and the pathogen was identified and confirmed as D. fangzhongdai. Here we report that D. fangzhongdai causes soft rot of P. ternata in China. The disease progressed very rapidly, and reduced the yield and quality of tubers. Thus, more research is needed to implement effective strategies to manage this disease.

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First Report of Bacterial Soft Rot Caused by Pantoea agglomerans on Chinese yam (Dioscorea opposita Thunb.) in China

Plant Disease ◽

10.1094/pdis-10-21-2151-pdn ◽

2022 ◽

Author(s):

Susu Fan ◽

Fangyuan Zhou ◽

Xueying Xie ◽

Xinjian Zhang ◽

Tielin Wang

Keyword(s):

16S Rdna ◽

Soft Rot ◽

Henan Province ◽

Pantoea Agglomerans ◽

Bacterial Suspension ◽

Pathogenicity Test ◽

Bacterial Soft Rot ◽

First Report ◽

Chinese Yam ◽

Dioscorea Opposita

Chinese yam (Dioscorea opposita Thunb.), which belongs to the family of Dioscorea, is widely naturalized throughout China, due to its high economic and medicinal value. Since 2019, water-soaked lesions were frequently observed in the underground tubers of Chinese yam located in Xinyang City, Henan Province. To identify the causal agent, ten pieces of tissue from the underground tubers with disease symptoms were collected. Those infected tissues (5×5 mm) were crushed in 500 μL sterilized water after surface sterilization and streaked onto Luria-Bertani agar plates. Pale-yellowish, rod-shaped, slimy single bacterial colonies with smooth margin were observed after 24 hours of incubation, and three bacterial colonies (named CY-1, CY-2 and CY-3) were randomly selected for further biochemical and molecular characterization. These bacteria were gram-negative with the cell length of 1.0 to 3.0 μm, width of 0.5 to 1.0 μm, and with peritrichous flagella. Subsequently, the bacteria were biochemically analyzed through BIOLOG (Hayward, CA) and identified as Pantoea agglomerans with 99% probability. Furthermore, the phylogenetic analysis results based on 16S rDNA, DNA gyrase subunit B (gyrB), and RNA polymerase sigma factor (rpoD) showed these three isolates were most closely related to P. agglomerans. The sequence of 16S rDNA, gyrB and rpoD of each strain was submitted to GenBank with the accession numbers MZ541065 MZ541066 and MZ541067 for 16S rDNA; MZ669846, MZ669847 and MZ669848 for gyrB; MZ669849, MZ669850 and MZ669851 for ropD. Pathogenicity test was performed to complete Koch’s postulates. Tubers of Chinese yam were wounded by sterile needle and inoculated with 500 μL 108 CFU/mL bacterial suspension. Sterilized water was used as a control. Five pots were inoculated for each isolate. Water-soaked lesions appeared after five days incubation at 25°C in a biochemical incubator and no lesions were observed on the control. Bacteria re-isolated from the lesions were similar in phenotypic and molecular characteristics to the original isolates. In brief, based on colony morphology, biochemical tests, characteristic sequence analysis, and pathogenicity verification, the pathogen responsible for the soft rot of Chinese yam in Henan Province was identified as P. agglomerans. In China, P. agglomerans has been reported to associate with bacterial soft rot on Chinese cabbage (Guo et al., 2020). To our knowledge, this work is the first report of bacterial rot caused by P. agglomerans on Chinese yam.

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

Plant Disease ◽

10.1094/pdis-06-21-1199-pdn ◽

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.

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First report of bacterial shot-hole disease caused by Xanthomonas arboricola pv. pruni on plumcot in South Korea

Plant Disease ◽

10.1094/pdis-06-20-1227-pdn ◽

2020 ◽

Author(s):

Walftor Dumin ◽

Mi-Jeong Park ◽

Jong-Han Park ◽

Chang Youl Yang ◽

Chang-Gi Back

Keyword(s):

16S Rdna ◽

Causal Agent ◽

Bacterial Suspension ◽

Pathogenicity Test ◽

Total Production ◽

Sterile Water ◽

First Report ◽

Young Leaves ◽

Leaf Tissues ◽

Xanthomonas Arboricola

Plumcot is an interspecific hybrid product between Japanese plums (Prunus salicina) and apricots (Prunus armeniaca) obtained by the NIHHS, Korea in 1999 [1]. At the early of 2017, black spots-like symptoms were observed on plumcot fruits and leaves at cultivation areas in Naju (34.965595, 126.665853) province. Further investigation shows that approximately 60% of the plumcot leaves in the affected orchard were infected, which caused 40% total production loss. At the early stage of infection, disease symptoms appear as small, angular and water-soaked spots and develop into circular brown spots at the later stages of infection. As the disease progresses, the leaf tissues around the spots became yellow and the lesions enlarged. When the adjacent lesions merged and the necrotic tissues fall off, shot-hole symptoms appear on the leaves. To identify the causal agent of this disease, infected leaf tissues were excised and surface-sterilized with 1% NaOCl for 30 secs prior to rinsing with sterile water, thrice . Tissue samples were then placed in sterile water (0.5 mL) for 5 min before its aliquots were streaked onto Luria-Bertani (LB) agar. Plates then were incubated at 28°C. To obtain pure colonies, bacteria were re-streak into a new LB agar and colonies showing typical Xanthomonas spp. morphology (i.e. convex, smooth, yellow, and mucoid) were subjected to Gram staining assay. For molecular identification, 16S ribosomal DNA (16S-rDNA) and gyrase B (gyrB) genes were amplified using a 9F/1512r and UP-1/UP-2Sr primers [2,3] respectively from 5 gram-negative isolates. PCR products were sequenced and analysed using BLASTN. Result shows that 16S-rDNA and gyrB genes are 99-100% identical to a similar genomic region of Xanthomonas arboricola pv. pruni (Xap) isolated in almond (MK156163), peach (MG049922) and apricot (KX950802) respectively [4,5,6]. 16S-rDNA and gyrB gene sequences were deposited in the GenBank (LC485472 and LC576824), whereas pathogen isolate was deposited into Korean Agricultural Culture Collection (KACC19949). Pathogenicity test was performed using Xap bacterial suspension (108 cfu/mL) inoculated on the abaxial and adaxial surface of plumcot detached leaves. For inoculation, 10 healthy young leaves were used whereas, 5 young leaves mock-inoculated with sterile LB broth were used as a control. Both leaf samples were kept in a closed container to maintain 100% humidity before being incubated at 25°C. The water-soaked symptoms were observed visually on the inoculated leaves 2 to 3 days post-inoculation. No water-soaked symptoms were observed on the control leaves. Morphology and sequences of molecular markers used showed that the 3 bacterial colonies re-isolated from the inoculated leaves were identical to the original isolate, fulfilling Koch’s postulate. Pathogenicity tests were repeated twice and the results obtained were consistent with the first experiment. As a new variety of stone fruit cultivated in Korea, information about pathogens and registered agrochemicals to control disease outbreak in plumcot are still limited. Therefore, the identification of Xap as a causal agent to the black spot disease is critical for the development of disease management strategies and to identify appropriate agrochemicals to control the occurrence of this disease in the field. To our knowledge, this is the first report of Xap as a causal agent to the shot-hole disease on the plumcot in Korea.

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

Plant Disease ◽

10.1094/pdis-02-21-0316-pdn ◽

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.

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