First Report of Pectobacterium polaris Causing Aerial Stem Rot of Potato in China

In July 2020, potato plants (cv. Xisen 6) showing characteristic symptoms of aerial stem rot were observed in a field in Fengning Manchu Autonomous County, Chengde, Hebei Province (northern China). The disease incidence in that field (5 ha in size) was more than 50%. Aerial stem rot of potato has increased in prevalence over recent years in Chengde, it can cause significant yield loss on susceptible cultivars such as Xisen 6 and Huangxin 226. Affected stem (light brown and water-soaked stem sections) pieces ca. 0.5 cm in length were surface-sterilized by dipping them in 75% ethanol for one min and then three successive rinses with sterile distilled water. Then, the tissues were soaked in 200 µl 0.9% saline for 20 min. Aliquots (20 μl) of three tenfold dilutions of the tissue specimen soaking solution were plated onto the crystal violet pectate (CVP) medium. The CVP plates were incubated at 28°C for 48 h. Colonies producing pits were restreaked and purified on Luria-Bertani (LB) agar plates. The bacterial gDNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech, Beijing, China). The 16S rDNA region was amplified by PCR using the universal primers 27F/1492R (Weisburg et al. 1991) and sequenced. Results of the Blastn analysis of the 16S rDNA amplicons (MZ348607, MZ348608) suggested that the isolates FN20211 and FN20222 belonged to the genus Pectobacterium. Housekeeping genes including acnA, gapA, icdA, mdh, proA and rpoS were also amplified using a set of primers (Ma et al. 2007; Waleron et al. 2008) followed by sequencing (MZ356250-MZ356261). To determine the species of the stem rot Pectobacterium isolates, multi-locus sequence analysis (MLSA) was performed with six housekeeping genes, and phylogenetic tree was reconstructed using RAxML (github.com/stamatak/standard-RAxML). No sequence variation was observed at any MLSA locus between FN20211 and FN20222. The result of phylogenetic analysis showed that the isolates clustered with P. polaris type strain NIBIO1006T, which was isolated from potato (Dees et al. 2017). And the concatenated sequence of the six loci of isolate FN20211/FN20222 is 100% identical to those of the strains PZ1 (CP046377.1) and WBC1 (GCF_011378945.1), which were isolated from potato in South Korea and from Chinese cabbage in China, respectively. Potato seedlings (cv. Xisen 6 and Favorita) were inoculated with the isolates FN20211 and FN20222 by injecting 100 µl of bacterial suspensions (108 CFU·mL-1) into the upper parts of the stems of potato plants, or injected with 100 µl of 0.9% saline as control. The seedlings were grown at 25°C and 50% relative humidity. Three days after inoculation, only the bacteria-inoculated seedlings showed disease symptoms resembling to those observed in the field. Bacterial colonies were obtained from the infected stems and were identified using the same PCR primers as described above. Therefore, P. polaris isolates FN20211 and FN20222 fulfill Koch’s postulates for aerial stem rot of potato. P. polaris causing blackleg and soft rot on potato plants has been reported in European countries including Netherlands, Norway (Dees et al. 2017) and Poland (Waleron et al. 2019), and also in Pakistan (Sarfraz et al. 2019) and Russia (Voronina et al. 2021). To our knowledge, this is the first report of P. polaris causing aerial stem rot of potato in China. The stem rot poses a significant threat to the local potato industry, and further research on epidemiology and disease management options is needed.

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First Report of Pectobacterium versatile Causing Aerial Stem Rot of Potato in China

Plant Disease ◽

10.1094/pdis-06-21-1264-pdn ◽

2021 ◽

Author(s):

Wanxin Han ◽

Jinhui Wang ◽

Zheng Li ◽

Yang Pan ◽

Dai Zhang ◽

...

Keyword(s):

16S Rdna ◽

Disease Incidence ◽

New York State ◽

Housekeeping Genes ◽

Soft Rot ◽

Stem Rot ◽

Post Inoculation ◽

First Report ◽

Potato Plants ◽

Aerial Stem

Pectobacterium species cause blackleg, soft rot and stem rot in potato and many other vegetable crops (Charkowski 2015). In July 2020, potato plants showing characteristic symptoms of aerial stem rot were observed in a field (cv. Xisen 6) in Fengning Manchu Autonomous County, Chengde, Hebei Province (North China). The disease incidence in that field (5 ha in size) was more than 50%. Putative pectolytic bacteria were obtained from symptomatic stem tissues (light brown and water-soaked stem sections) by culturing on the crystal violet pectate (CVP) medium. Bacterial colonies producing pits, were restreaked and purified on Luria-Bertani (LB) agar. The isolates causing stem rot were gram negative and rod shaped, negative for oxidase, urease, indole production, gelatin liquefaction and acid production from maltose and D-sorbitol. All isolates were catalase positive, produced acid from lactose, rhamnose, saccharose, raffinose and D-arabinose, and were tolerant to 5% NaCl, and able to utilize citrate. The bacterial gDNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech). The 16S rDNA region was amplified by PCR using the universal primer pair 27F/1492R and sequenced. Result of the Blastn analysis of the 16S rDNA amplicons (MZ379788, MZ379789) suggested that the isolates FN20111 and FN20121 belonged to the genus Pectobacterium. To determine the species of the stem rot Pectobacterium isolates, multi-locus sequence analysis (MLSA) was performed with six housekeeping genes acnA, gapA, icdA, mdh, proA and rpoS (MZ403781-MZ403792), and phylogenetic tree was reconstructed using RAxML v8.2.12 (https://github.com/stamatak/standard-RAxML). The result of phylogenetic analysis showed that the stem rot Pectobacterium isolates FN20111 and FN20121 clustered with P. versatile (syn. ‘Candidatus Pectobacterium maceratum’) strains CFBP6051T (Portier et al. 2019), SCC1 (Niemi et al. 2017) and F131 (Shirshikov et al. 2018). And the isolates FN20111 and FN20121 were more closely related to the type strain CFBP6051T than to strains SCC1 and F131. Potato seedlings (cv. Xisen 6 and Favorita) were inoculated with the isolates FN20111 and FN20121 by injecting 100 µl of bacterial suspensions (108 CFU·mL-1) into the upper parts of the stems of potato plants, or injected with 100 µl of 0.9% saline solution as control. The seedlings were grown at 28°C and 50% relative humidity. Three days post-inoculation, only the bacteria-inoculated seedlings showed diseased symptoms resembling to those observed in the field. Bacterial colonies were obtained from the infected stems and were identified using the same PCR primers of housekeeping genes as described above, fulfill Koch’s postulates. P. versatile causing soft rot and blackleg on potato plants has been reported in Finland (Niemi et al. 2017), Russia (Shirshikov et al. 2018), Netherlands (Portier et al. 2019), Poland (Waleron et al. 2019) and in New York State (Ma et al. 2021). To our knowledge, this is the first report of P. versatile causing aerial stem rot of potato in China.

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First Report of Rhizome Rot Caused by Pectobacterium brasiliense on Ginger in China

Plant Disease ◽

10.1094/pdis-10-21-2324-pdn ◽

2022 ◽

Author(s):

Jinhui Wang ◽

Yuxiang Lu ◽

Wanxin Han ◽

Lijun Fu ◽

Xiaoqing Han ◽

...

Keyword(s):

16S Rdna ◽

Disease Incidence ◽

Housekeeping Genes ◽

Zingiber Officinale ◽

Soft Rot ◽

Management Options ◽

Universal Primer ◽

First Report ◽

Wide Range ◽

Rhizome Rot

In August 2020, ginger (Zingiber officinale) rhizomes (cv. Mianjiang) showing soft rot symptoms were observed in a field in Tayang Village, Fengrun District, Tangshan, Hebei Province (North China). The disease incidence in that field (15 ha in size) was more than 20%. Symptomatic rhizomes (brown and water-soaked) were surface-sterilized in 75% ethanol for 60 sec and then three successive rinses with sterile distilled water. Rhizomes were cut into pieces ca. 0.5 cm in length, and then were soaked in 500 µl 0.9% saline for 20 min. Aliquots (20 μl) of three tenfold dilutions of the tissue specimen soaking solution were plated onto the lysogeny broth (LB) medium. And LB plates were incubated at 28°C for 24 h. Five single colonies were picked from each LB plate and restreaked three times for purity. Endophytic bacteria were also isolated from asymptomatic rhizomes as control. The bacterial gDNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech, Beijing, China). The 16S rDNA region was amplified by PCR using the universal primer pair 27F/1492R (Weisburg et al. 1991) and sequenced. The results of BLASTN against NCBI nr of the 16S rDNA amplicons suggested that the most isolates (8/10) obtained from the rotten rhizomes belonged to the genus Pectobacterium, and few isolates (2/10) were Enterobacter spp.. Only Enterobacter spp. were isolated from asymptomatic rhizomes. Since all Pectobacterium isolates showed identical 16S rDNA sequence, thus, only two isolates were selected for further analysis. Pectobacterium isolates TS20HJ1 and TS20HJ2 (MZ853520, MZ853521) represent isolates from two plant individuals. To determine the species of the rhizome rot Pectobacterium isolates, multi-locus sequence analysis (MLSA) was performed with five housekeeping genes acnA, icdA, mdh, proA and rpoS (MZ994717-MZ994726) (Ma et al. 2007; Waleron et al. 2008), and a phylogenetic tree was reconstructed using RAxML v8.2.12 (github.com/stamatak/standard-RAxML). No sequence variation was observed at any MLSA locus between the two isolates. The result of phylogenetic analysis showed that the ginger rhizome isolates clustered with P. brasiliense type strain IBSBF1692T (Duarte et al. 2004; Nabhan et al. 2012). Ginger seedlings (cv. Mianjiang) were inoculated with the isolate TS20HJ1 by injecting 10 µl of bacterial suspensions (108 CFU·mL-1) into the rhizomes, or injected with 10 µl of 0.9% saline solution as control. The seedlings were grown at 28°C and 50% relative humidity. Ten days after inoculation, only the bacteria-inoculated rhizomes showed diseased symptoms resembling to those observed in the field. Bacterial colonies were obtained from the infected rhizomes and were identified with MLSA gene sequencing, fulfilling Koch’s postulates. P. brasiliense causes soft rot of a wide range of economically important crops (Oulghazi et al. 2021). To our knowledge, this is the first report of P. brasiliense causing rhizome rot of ginger in China. The rhizome rot caused 20-25% yield loss on average in Tangshan region in 2020, which poses a significant threat to the local ginger farming. Further research on epidemiology and disease management options is needed.

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First Report of a 16SrIX Group (Pigeon Pea Witches'-Broom) Phytoplasma Associated with Sesame Phyllody in Turkey

Plant Disease ◽

10.1094/pdis-11-12-1100-pdn ◽

2013 ◽

Vol 97 (6) ◽

pp. 835-835 ◽

Cited By ~ 12

Author(s):

M. Catal ◽

C. Ikten ◽

E. Yol ◽

R. Üstün ◽

B. Uzun

Keyword(s):

16S Rdna ◽

Nested Pcr ◽

Disease Incidence ◽

Natural Infection ◽

Reference Sequence ◽

Universal Primers ◽

Flower Formation ◽

Disease Symptoms ◽

First Report ◽

Rflp Profile

Sesame (Sesamum indicum L.) is an important oilseed crops widely grown in the southern regions of Turkey. Sesame seeds are primarily used in production of tahini as well as a garnish on sweets and bakery products in the country. Sesame plants with phyllody disease symptoms have increasingly been observed in the fields of Antalya province since 2007. The disease incidence in these fields was found to range from 37 to 62% (2). Infected plants display a variety of the disease symptoms such as virescence, asymptomatic shoot proliferation, infertile flower formation, reduced leaf size, and thin and weak capsule development. Total genomic DNA was extracted from samples collected from symptomatic (10 plants) and asymptomatic healthy-looking plants (10 plants) using a CTAB method and amplified with universal primers P1/P7 and R16F2n/R16R2 in direct and nested PCR, respectively (1,3). Amplifications of the DNA from the symptomatic plants yielded a product of 1.8 kb in direct and 1.2 kb in nested PCR assays. No amplification was observed in symptomless plants of the same age and collected from the same fields. Amplicons were purified, cloned in a pTZ57R/T Vector, and sequenced using a Beckman Coulter 8000 CEQ Genetic Analysis System. Four aligned 16S rDNA sequences (1,845 bp) were found to be all identical and belonging to one species. One sequence was deposited in GenBank under the accession number KC139791. A BLAST similarity search revealed that the sequence shared 99% homology with the sequences of the members of 16SrIX group phytoplasmas, ‘Brassica rapa’ phyllody phytoplasma (HM559246.1) and Iranian Almond witches'-broom phytoplasma (DQ195209.1) available in GenBank. In addition, iPhyClassifier software (4) was employed to create a virtual RFLP profile. The analysis showed that the RFLP profile of the sesame phytoplasma 16S rDNA sequence is identical (a similarity coefficient of 1.00) to the profile of the 16Sr group IX phytoplasma reference sequence (Y16389). A phylogenetic tree was also constructed using the neighbor joining plot option of the Clustal X program. The sequence clustered together with 16SrIX group phytoplasmas. To our knowledge, this is the first report of a natural infection of sesame by a new phytoplasma species from the 16SrIX group in Turkey. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) C. Ikten et al. Phytopathogenic Mollicutes 1:101, 2011. (3) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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First Report of Group 16SrXII Phytoplasma Causing Stolbur Disease in Potato Plants in the Eastern and Southern Anatolia Regions of Turkey

Plant Disease ◽

10.1094/pdis-06-10-0439 ◽

2010 ◽

Vol 94 (11) ◽

pp. 1374-1374 ◽

Cited By ~ 6

Author(s):

S. Eroglu ◽

H. Ozbek ◽

F. Sahin

Keyword(s):

16S Rdna ◽

Rdna Sequence ◽

Universal Primers ◽

Effective Control ◽

Diseased Plant ◽

Eastern Anatolia ◽

Universal Primer ◽

16S Rdna Sequence ◽

First Report ◽

Potato Plants

In recent years, a stolbur-like disease has had devastating effects on the yield and marketable quality of potato production in Erzurum (Eastern Anatolia) and Akcakale-Sanliurfa (Southern Anatolia) regions of Turkey. Potato plants exhibited several different symptoms including stunting, upward rolling of the top leaves along with reddish or purplish coloration, chlorosis, shortened internodes, swollen nodes, proliferated axillary buds, aerial tubers, and early plant decline. An extensive survey from 2003 to 2010 was performed and diseased plant samples were collected. Total genomic DNAs were isolated from the leaf mid-veins of the six different symptomatic and two symptomless plants selected. Nested-PCRs, carried out by using phytoplasma-universal primer pair P1/P7 followed by R16F2n/R16R2 (2), amplified 16S rDNA fragments (F2nR2) from only templates derived from symptomatic plants. F2nR2 PCR products from two independent symptomatic plants were cloned and sequenced from both directions with M13 universal primers. The obtained 16S rDNA sequence (GenBank Accession No HM485579) was subjected to virtual restriction fragment length polymorphism (RFLP) analysis using iphyclassifier software (3). Results indicated that the phytoplasma, here identified in association with potato plants, shared best sequence identity (99%) with members of subgroup 16SrXII-A (e.g., GenBank Accession No. EU010006). Moreover, collective RFLP pattern of potato-associated phytoplasma differed from digestion profiles of previously described 16SrXII subgroups, sharing best similarity coefficient (0.94) with the reference phytoplasma strain of subgroup 16SrXII-A (GenBank Accession No. AJ964960). Thus, it was confirmed that potato-associated phytoplasma represents a new 16SrXII subgroup (16SrXII-N). Furthermore, a new primer set (PatsecF/PatsecR) was designed for priming specific PCR-amplification of potato-associated phytoplasma 16S rDNA sequence. PCR reaction was successfully used for specifically detecting stolbur phytoplasma in infected potato plants. The use of this method may help to determine possible alternative hosts and vectors of potato phytoplasma, which is important for development of an integrated management strategy for effective control of this disease in the future. Presence of potato stolbur diseases in the Eastern Anatolia Region of Turkey has previously been reported (1). To our knowledge, this is the first report of occurrence of a 16SrXII group phytoplasma causing potato stolbur diseases caused in the Eastern and Southern Anatolia regions of Turkey. References: (1) A. Citir. J. Turk. Phytopathol. 14:53, 1985. (2) D. E. Gundersen and I. M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3)Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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First Report of Stalk Rot of Celery Caused by Erwinia rhapontici in China

Plant Disease ◽

10.1094/pdis-07-21-1364-pdn ◽

2021 ◽

Author(s):

Jinhui Wang ◽

Wanxin Han ◽

Yang Pan ◽

Aiguo Guo ◽

Dai Zhang ◽

...

Keyword(s):

16S Rdna ◽

Prunus Persica ◽

Housekeeping Genes ◽

Soft Rot ◽

Pcr Primers ◽

Apium Graveolens ◽

Management Options ◽

Disease Symptoms ◽

First Report ◽

Stalk Rot

Species belonging to the genus Erwinia cause diseases in many economically important plants (Mansfield et al. 2012). In May 2021, celery plants (Apium graveolens var. dulce) showing soft rot symptoms were observed in greenhouses (cv. Queen of France) in Boye County, Baoding, Hebei Province (North China). Disease symptoms began with pinkish water-soaked lesions on the midrib of celery stalks, but at the same time the leaves and root did not show symptoms. The infected celery plants rapidly developed brownish rotten stalks and leaves turned dry and yellow, but root remained asymptomatic. The disease incidence in two greenhouses (0.15 ha in size) was more than 50%. Affected celery stalk tissues were cut into 0.5 cm pieces, followed by surface sterilization using 75% ethanol for 60 sec and then three successive rinses with sterile distilled water. Then, the tissues were immersed in 200 µl 0.9% saline for 15 min. Aliquots of two tenfold dilutions of the tissue specimen soaking solution were plated onto Luria-Bertani (LB) agar plates and incubated at 28°C for 24 h. Single colonies were picked and restreaked onto LB agar three times for purity. The bacterial gDNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech). The 16S rDNA region was amplified by PCR using the universal primers 27F/1492R and sequenced. Result of blastn analysis of the 16S rDNA amplicons (MZ489246-MZ489247) indicated that the bacterial isolates (BY21311 and BY21312) belonged to the genus Erwinia. Biolog analysis (GEN III Microplate) identified the two isolates BY21311 (SIM=0.668) and BY21312 (SIM=0.638) as E. rhapontici. Housekeeping genes including acnA, gapA, icdA, mdh and rpoS were also amplified using a set of PCR primers (Ma et al. 2007; Waleron et al. 2008) followed by sequencing (MZ463029-MZ463038). To determine the species of the Erwinia isolates BY21311 and BY21312, multi-locus sequence analysis (MLSA) was performed with five housekeeping genes, and phylogenetic tree was reconstructed using RAxML v8.2.12 (Stamatakis et al. 2005). No sequence variation was observed at any MLSA locus between BY21311 and BY21312. The result of phylogenetic analysis showed that the celery stalk rot isolates BY21311 and BY21312 were clustered with E. rhapontici isolates. These celery isolates are closely related to the cabbage (Brassica rapa) isolate MAFF311153 (AP024329.1) in Japan. When celery plants have eight to nine true leaves, plants (cv. Queen of France) were inoculated with the isolate BY21311 by injecting 20 µl of bacterial suspensions (106 CFU·mL-1) into the celery stalks, or injected with 20 µl of 0.9% saline as control. The seedlings were grown at 25 °C and 50% relative humidity. Three days after inoculation, only infected seedling showed disease symptoms resembled to those observed in greenhouses. Bacterial colonies were obtained from the infected stalks and were identified using the same PCR primers of housekeeping genes as described above, fulfill Koch’s postulates. E. rhapontici has been reported to cause pink seed, crown and stem rot, soft rot or leaf spot on many plant hosts including pea (Pisum sativum), chickpea (Cicer arietinum), lentil (Lens culinaris), common bean (Phaseolus vulgaris), lucerne (Medicago sativa), wheat (Triticum aestivum), hyacinth (Hyacinthus orientalis), onion (Allium cepa), kiwifruit (Actinidia chinensis) and peach (Prunus persica) (Huang et al. 2003; Wang et al. 2017; Zhang et al. 2018; Kovács et al. 2020). To our knowledge, this is the first report of E. rhapontici causing stalk rot in celery. Stalk rot of celery has increased in prevalence over recent years in the Baoding region, it can cause significant yield loss and no cultivar has been found to be resistant to this disease so far. The stalk rot poses significant threat to local celery production, and further research on epidemiology and disease management options is needed.

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First Report of Alternaria Leaf Spot Caused by Alternaria sp. on Highbush Blueberry (Vaccinium corymbosum) in South Korea

Plant Disease ◽

10.1094/pdis-04-14-0344-pdn ◽

2014 ◽

Vol 98 (10) ◽

pp. 1434-1434

Author(s):

J.-H. Kwon ◽

D.-W. Kang ◽

M.-G. Cheon ◽

J. Kim

Keyword(s):

South Korea ◽

Leaf Spot ◽

Disease Incidence ◽

Vaccinium Corymbosum ◽

Its Rdna ◽

Universal Primers ◽

Conidial Suspension ◽

First Report ◽

Representative Isolate ◽

Alternaria Sp

In South Korea, the culture, production, and consumption of blueberry (Vaccinium corymbosum) have increased rapidly over the past 10 years. In June and July 2012, blueberry plants with leaf spots (~10% of disease incidence) were sampled from a blueberry orchard in Jinju, South Korea. Leaf symptoms included small (1 to 5 mm in diameter) brown spots that were circular to irregular in shape. The spots expanded and fused into irregularly shaped, large lesions with distinct dark, brownish-red borders. The leaves with severe infection dropped early. A fungus was recovered consistently from sections of surface-disinfested (1% NaOCl) symptomatic leaf tissue after transfer onto water agar and sub-culture on PDA at 25°C. Fungal colonies were dark olive and produced loose, aerial hyphae on the culture surfaces. Conidia, which had 3 to 6 transverse septa, 1 to 2 longitudinal septa, and sometimes also a few oblique septa, were pale brown to golden brown, ellipsoid to ovoid, obclavate to obpyriform, and 16 to 42 × 7 to 16 μm (n = 50). Conidiophores were pale to mid-brown, solitary or fasciculate, and 28 to 116 × 3 to 5 μm (n = 50). The species was placed in the Alternaria alternata group (1). To confirm the identity of the fungus, the complete internal transcribed spacer (ITS) rDNA region of a representative isolate, AAVC-01, was amplified using ITS1 and ITS4 primers (2). The DNA products were cloned into the pGEM-T Easy vector (Promega, Madison, WI) and the resulting pOR13 plasmid was sequenced using universal primers. The resulting 570-bp sequence was deposited in GenBank (Accession No. KJ636460). Comparison of ITS rDNA sequences with other Alternaria spp. using ClustalX showed ≥99% similarity with the sequences of A. alternata causing blight on Jatropha curcas (JQ660842) from Mexico and Cajannus cajan (JQ074093) from India, citrus black rot (AF404664) from South Africa, and other Alternaria species, including A. tenuissima (WAC13639) (3), A. lini (Y17071), and A. longipes (AF267137). Two base substitutions, C to T at positions 345 and 426, were found in the 570-bp amplicon. Phylogenetic analysis revealed that the present Alternaria sp. infecting blueberry grouped separately from A. tenuissima and A. alternata reported from other hosts. A representative isolate of the pathogen was used to inoculate V. corymbosum Northland leaves for pathogenicity testing. A conidial suspension (2 × 104 conidia/ml) from a single spore culture and 0.025% Tween was spot inoculated onto 30 leaves, ranging from recently emerged to oldest, of 2-year-old V. corymbosum Northland plants. Ten leaves were treated with sterilized distilled water and 0.025% Tween as a control. The plants were kept in a moist chamber with >90% relative humidity at 25°C for 48 h and then moved to a greenhouse. After 15 days, leaf spot symptoms similar to those observed in the field developed on the inoculated leaves, whereas the control plants remained asymptomatic. The causal fungus was re-isolated from the lesions of the inoculated plants to fulfill Koch's postulates. To our knowledge, this is the first report of Alternaria sp. on V. corymbosum in South Korea. References: (1) E. G. Simmons. Page 1797 in: Alternaria: An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, The Netherlands, 2007. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990. (3) M. P. You et al. Plant Dis. 98:423, 2014.

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First report of banana (Musa acuminate L. AAA Cavendish, cv. Formosana) leaf spot disease caused by Curvularia geniculata in China

Plant Disease ◽

10.1094/pdis-09-21-2001-pdn ◽

2021 ◽

Author(s):

Yanxiang Qi ◽

Yanping Fu ◽

Jun Peng ◽

Fanyun Zeng ◽

Yanwei Wang ◽

...

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Universal Primers ◽

Leaf Spot Disease ◽

Leaf Margin ◽

Conidial Suspension ◽

Tropical Fruit ◽

First Report ◽

Spot Disease

Banana (Musa acuminate L.) is an important tropical fruit in China. During 2019-2020, a new leaf spot disease was observed on banana (M. acuminate L. AAA Cavendish, cv. Formosana) at two orchards of Chengmai county (19°48ʹ41.79″ N, 109°58ʹ44.95″ E), Hainan province, China. In total, the disease incidence was about 5% of banana trees (6 000 trees). The leaf spots occurred sporadically and were mostly confined to the leaf margin, and the percentage of the leaf area covered by lesions was less than 1%. Symptoms on the leaves were initially reddish brown spots that gradually expanded to ovoid-shaped lesions and eventually become necrotic, dry, and gray with a yellow halo. The conidia obtained from leaf lesions were brown, erect or curved, fusiform or elliptical, 3 to 4 septa with dimensions of 13.75 to 31.39 µm × 5.91 to 13.35 µm (avg. 22.39 × 8.83 µm). The cells of both ends were small and hyaline while the middle cells were larger and darker (Zhang et al. 2010). Morphological characteristics of the conidia matched the description of Curvularia geniculata (Tracy & Earle) Boedijn. To acquire the pathogen, tissue pieces (15 mm2) of symptomatic leaves were surface disinfected in 70% ethanol (10 s) and 0.8% NaClO (2 min), rinsed in sterile water three times, and transferred to potato dextrose agar (PDA) for three days at 28°C. Grayish green fungal colonies appeared, and then turned fluffy with grey and white aerial mycelium with age. Two representative isolates (CATAS-CG01 and CATAS-CG92) of single-spore cultures were selected for molecular identification. Genomic DNA was extracted from the two isolates, the internal transcribed spacer (ITS), large subunit ribosomal DNA (LSU rDNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1-α) and RNA polymerase II second largest subunit (RPB2) were amplified and sequenced with universal primers ITS1/ITS4, LROR/LR5, GPD1/GPD2, EF1-983F/EF1-2218R and 5F2/7cR, respectively (Huang et al. 2017; Raza et al. 2019). The sequences were deposited in GenBank (MW186196, MW186197, OK091651, OK721009 and OK491081 for CATAS-CG01; MZ734453, MZ734465, OK091652, OK721100 and OK642748 for CATAS-CG92, respectively). For phylogenetic analysis, MEGA7.0 (Kumar et al. 2016) was used to construct a Maximum Likelihood (ML) tree with 1 000 bootstrap replicates, based on a concatenation alignment of five gene sequences of the two isolates in this study as well as sequences of other Curvularia species obtained from GenBank. The cluster analysis revealed that isolates CATAS-CG01 and CATAS-CG92 were C. geniculata. Pathogenicity assays were conducted on 7-leaf-old banana seedlings. Two leaves from potted plants were stab inoculated by puncturing into 1-mm using a sterilized needle and placing 10 μl conidial suspension (2×106 conidia/ml) on the surface of wounded leaves and equal number of leaves were inoculated with sterile distilled water serving as control (three replicates). Inoculated plants were grown in the greenhouse (12 h/12 h light/dark, 28°C, 90% relative humidity). Necrotic lesions on inoculated leaves appeared seven days after inoculation, whereas control leaves remained healthy. The fungus was recovered from inoculated leaves, and its taxonomy was confirmed morphologically and molecularly, fulfilling Koch’s postulates. C. geniculata has been reported to cause leaf spot on banana in Jamaica (Meredith, 1963). To our knowledge, this is the first report of C. geniculata on banana in China.

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First Report on the Molecular Identification of Phytoplasma (16SrII-D) Associated with Witches' Broom of Kalmegh (Andrographis paniculata) in India

Plant Disease ◽

10.1094/pdis-08-14-0854-pdn ◽

2015 ◽

Vol 99 (1) ◽

pp. 155-155 ◽

Cited By ~ 3

Author(s):

S. T. Saeed ◽

A. Khan ◽

A. Samad

Keyword(s):

16S Rdna ◽

Nested Pcr ◽

Disease Incidence ◽

Antioxidant Properties ◽

First Record ◽

Andrographis Paniculata ◽

Medicinal And Aromatic Plants ◽

First Report ◽

16S Rdna Sequence Analysis ◽

Virtual Rflp

Andrographis paniculata (family Acanthaceae), also known as “King of Bitters” or Kalmegh, is an important medicinal plant used for the treatment of various diseases. It has antimicrobial, antiviral, anti-inflammatory, hepatoprotective, antidiabetic, antihyperglycemic, and antioxidant properties (1). During June 2014, while performing a routine survey of the commercial trial fields of Kalmegh at Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, India, typical phytoplasma disease symptoms such as virescence, proliferation, and witches' broom along with little leaf and stunted growth were observed. The disease incidence was estimated to be approximately 7 to 10%. To ascertain the presence of phytoplasma, 16 samples of leaves were collected from nine different field sites, and total genomic DNA was extracted from the symptomatic and symptomless Kalmegh plants by the CTAB method. Direct and nested PCR assays were performed targeting the 16S rDNA using generic phytoplasma primer pairs P1/P6 followed by R16F2n/R16R2 (2). Resulting bands of the expected size (1.5 kb and 1.2 kb, respectively) were amplified from symptomatic plants. No amplification was observed with DNA from asymptomatic plant samples. The purified nested PCR products were cloned into E. coli DH5α, using the pGEM-T Easy vector (Promega, United States) and sequenced with primers M13For/M13Rev using an automatic sequencer (ABI Prism, Perkin Elmer) at CIMAP. The sequence was analyzed by BLASTn and found to share 99% similarity with Echinacea witches'-broom phytoplasma and Sesame phyllody phytoplasma strain (GenBank Accession Nos. JF340080 and KF612966, respectively), which belong to the 16SrII-D group. The sequence was deposited in NCBI as GenBank Accession No. KM359410. A phylogenetic tree using MEGA v5.0 (4) was constructed with 16S rDNA; consensus sequences of phytoplasmas belonging to distinct groups revealed that the present phytoplasma clustered with the 16SrII group. iPhyClassifier software was used to perform sequence comparison and generate a virtual restriction fragment length polymorphism (RFLP) profile (5). On the basis of iPhyClassifier, the 16S rDNA sequence analysis of our isolate showed 99.2% similarity with that of the ‘Candidatus Phytoplasma australasiae’ reference strain (GenBank Accession No. Y10097), which belongs to 16Sr group II. The virtual RFLP pattern of F2n/R2 fragment was most similar to the 16SrII-D subgroup (similarity coefficient of 0.91) but showed a difference in profile with HpaI, HhaI, and MseI enzymes. Several bacterial/fungal and viral diseases have been reported on A. paniculata (3); however, to our knowledge, this is the first report of witches' broom disease in India and the first record of a 16SrII-D group phytoplasma on Kalmegh. Its presence in Kalmegh is of great significance due to its commercial interest. References: (1) S. Akbar. Altern. Med. Rev. 16:1, 2011. (2) D. E. Gundersen and M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3) A. Khan and A. Samad. Plant Dis. 98:698, 2014. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011. (5) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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First Report of Candidatus Phytoplasma solani Associated with Potato Plants in Greece

Plant Disease ◽

10.1094/pdis-05-14-0488-pdn ◽

2014 ◽

Vol 98 (12) ◽

pp. 1739-1739 ◽

Cited By ~ 3

Author(s):

M. C. Holeva ◽

P. E. Glynos ◽

C. D. Karafla ◽

E. M. Koutsioumari ◽

K. B. Simoglou ◽

...

Keyword(s):

16S Rdna ◽

Nested Pcr ◽

Pathogenic Bacteria ◽

Related Strain ◽

Northern Greece ◽

First Report ◽

Potato Plants ◽

Pcr Product ◽

16S Rdna Pcr ◽

Stolbur Phytoplasma

In August 2013, potato plants (Solanum tuberosum) cv. Banba displaying symptoms resembling those caused by Candidatus Phytoplasma solani (potato stolbur phytoplasma) were observed in a 2-ha field in the area of the Peripheral Unit of Drama (northern Greece). The plants were 10 weeks old and their symptoms included reddening and upward rolling of leaflets, reduced size of leaves, shortened internodes, and aerial tuber formation. Incidence of affected plants was estimated to be 40% in the field. Four symptomatic potato plants were collected for laboratory testing of possible phytoplasma infection. From each of these four plants, total DNA was extracted from mid veins of reddish leaflets from apical shoot parts and of leaflets emerging from aerial tubers, using a phytoplasma enrichment procedure (1). A nested PCR using the phytoplasma universal 16S rRNA primer pairs: P1/P7 followed by R16F2n/R16R2 (3) amplified the expected ~1.2-kb 16S rDNA fragment in all four symptomatic potato plants. No amplification was observed with DNA similarly extracted from leaflets of asymptomatic potato plants of the same variety collected from an apparently healthy crop. One of the four 1.2-kb nested 16S rDNA PCR products was gel purified, cloned into the pGEM-T-easy plasmid vector (Promega, Madison, WI), and sequenced by Beckman Coulter Genomics (United Kingdom). At least twofold coverage per base position of the cloned PCR product was achieved. BLAST analysis showed that the obtained sequence of the PCR 16S rDNA product was: i) 100% identical to several GenBank sequences of Ca. P. solani strains, including strains detected previously in Greece infecting tomato (GenBank Accession No. JX311953) and Datura stramonium (HE598778 and HE598779), and ii) 99.7% similar to that of the Ca. P. solani reference strain STOL11 (AF248959). Furthermore, analysis by iPhyClassifier software showed that the virtual restriction fragment length polymorphism (RFLP) pattern of the sequenced PCR 16S rDNA product is identical (similarity coefficient 1.00) to the reference pattern of the 16SrXII-A subgroup (AF248959). The sequence of this PCR product was deposited in NCBI GenBank database under the accession no. KJ810575. The presence of the stolbur phytoplasma in all four symptomatic potato plants examined was further confirmed by nested PCR using the stolbur-specific STOL11 primers (3) targeting non-ribosomal DNA. Based on the observed symptoms in the field and laboratory molecular examinations, we concluded that the potato plants were infected by a Ca. P. solani related strain. The stolbur disease has been previously reported in Greece affecting tomato (2,5) and varieties of D. stramonium (4). To our knowledge, this is the first report of a Ca. P. solani related strain infecting a potato crop in Greece. As northern Greece is a center of potato production, the source of this pathogen is to be investigated. References: (1) U. Ahrens and E. Seemuller. Phytopathology 82:828, 1992. (2) A. S. Alivizatos. Pages 945-950 in: Proceedings of the 7th International Conference of Plant Pathogenic Bacteria. Academiai Kiado, Budapest, Hungary, 1989. (3) J. Jović et al. Bull. Insectol. 64:S83, 2011. (4) L. Lotos et al. J. Plant Pathol. 95:447, 2013. (5) E. Vellios and F. Lioliopoulou. Bull. Insectol. 60:157, 2007.

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First Report of Fusarium asiaticum Causing Stem Rot of Ligusticum chuanxiong in China

Plant Disease ◽

10.1094/pdis-05-21-1026-pdn ◽

2021 ◽

Author(s):

Tingting Zhu ◽

Linxuan Li ◽

Antonios Petridis ◽

George Xydis ◽

Maozhi Ren

Keyword(s):

Disease Incidence ◽

Elongation Factor ◽

Stem Rot ◽

Post Inoculation ◽

Pathogenicity Test ◽

First Report ◽

Ligusticum Chuanxiong ◽

Fusarium Asiaticum ◽

Sterile Needle ◽

Yellow Pigments

Ligusticum chuanxiong (known as Chuanxiong in China) is a traditional edible-medicinal herb, which has been playing important roles in fighting against COVID-19 (Ma et al. 2020). In March 2021, we investigated stem rot of Chuanxiong in six adjacent fields (~100 ha) in Chengdu, Sichuan Province, China. The disease incidence was above 5% in each field. Symptomatic plants showed stem rot, watersoaked lesions, and blackening with white hyphae present on the stems. Twelve symptomatic Chuanxiong plants (2 plants/field) were sampled. Diseased tissues from the margins of necrotic lesions were surface sterilized in 75% ethanol for 45 s, and 2% NaClO for 5 min. Samples were then rinsed three times in sterile distilled water and cultured on potato dextrose agar (PDA) at 25ºC for 72 h. Fourteen fungal cultures were isolated from 18 diseased tissues, of which eight monosporic isolates showed uniform characteristics. The eight fungal isolates showed fluffy white aerial mycelia and produced yellow pigments with age. Mung bean broth was used to induce sporulation. Macroconidia were sickle-shaped, slender, 3- to 5-septate, and averaged 50 to 70 μm in length. Based on morphological features of colonies and conidia, the isolates were tentatively identified as Fusarium spp. (Leslie and Summerell 2006). To identify the species, the partial translation elongation factor 1 alpha (TEF1-α) gene was amplified and sequenced (O’Donnell et al. 1998). TEF1-α sequences of LCSR01, LCSR02 and LCSR05 isolates (GenBank nos. MZ169386, MZ169388 and MZ169387) were 100%, 99.72% and 99.86% identical to that of F. asiaticum strain NRRL 26156, respectively. The phylogenetic tree based on TEF1-α sequences showed these isolates clustered with F. asiaticum using Neighbor-Joining algorithm. Furthermore, these isolates were identified using the specific primer pair Fg16 F/R (Nicholson et al. 1998). The results showed these isolates (GenBank nos. MZ164938, MZ164939 and MZ164940) were 100% identical to F. asiaticum NRRL 26156. Pathogenicity test of the isolate LCSR01 was conducted on Chuanxiong. After wounding Chuanxiong stalks and rhizomes with a sterile needle, the wounds were inoculated with mycelia PDA plugs. A total of 30 Chuanxiong rhizomes and stalks were inoculated with mycelia PDA plugs, and five mock-inoculated Chuanxiong rhizomes and stalks served as controls. After inoculation, the stalks and rhizomes were kept in a moist chamber at 25°C in the dark. At 8 days post inoculation (dpi), all inoculated stalks and rhizomes exhibited water-soaked and blackened lesions. At 10 dpi, the stalks turned soft and decayed, and abundant hyphae grew on the exterior of infected plants, similar to those observed in the field. No disease symptoms were observed on the control plants. The pathogen was re-isolated from the inoculated tissues and the identity was confirmed as described above. Ten fungal cultures were re-isolated from the 10 inoculated tissues, of which nine fungal cultures were F. asiaticum, fulfilling Koch’s postulates. To our knowledge, this is the first report of F. asiaticum causing stem rot of Chuanxiong in China. Chuanxiong has been cultivated in rotation with rice over multiple years. This rotation may have played a role in the increase in inoculum density in soil and stem rot epidemics in Chuanxiong. Diseased Chuanxiong may be contaminated with the mycotoxins produced by F. asciaticum, 3-acetyldeoxynivalenol or nivalenol, which may deleteriously affect human health. Therefore, crop rotations should be considered carefully to reduce disease impacts.

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