Yellow Colonies Research Articles

First Report of Xanthomonas euvesicatoria Causing Bacterial Leaf Spot of Pepper (Capsicum annuum) in Montenegro.

Leaf spot of pepper was observed on different pepper cultivars in central Montenegro during summer and early autumn in three consecutive growing seasons (2017 - 2019). Necrotic spots were numerous, varying in size, irregular in shape, brown, and surrounded by a weak halo. The most intensive symptoms were observed on lower leaves. In conditions conducive or the infection, the lesions merged resulting in the leaf drop. Symptoms were not observed on pepper stems and fruits. A total of seventeen bacterial strains were isolated from infected pepper leaves collected in seven different localities in the seasons of 2017-19. They formed yellow, convex, and mucoid colonies on yeast extract-dextrose-CaCO3 (YDC) medium and induced hypersensitive reaction in tobacco leaves. They were Gram negative, strictly aerobic, oxidase negative, catalase-positive, hydrolyzed gelatine and esculin and did not reduce nitrate, nor grew on 0.1% TTC and at 37°C. Out of tested 17 strains, eight hydrolyzed starch and three showed pectolytic activity, thus differing in these biochemical traits from Xanthomonas euvesicatoria (Xe) the reference strain KFB 1 (Obradović et al., 2004) used in all tests as a positive control. PCR analysis, with primer pair XeF/XeR, produced a single characteristic band of 173 bp in all 17 strains (Koenraadt et al. 2009). Additionally, the BOX-PCR profile of all the strains produced with the BOX A1R primer (Schaad et al. 2001) showed 100% homology with KFB 1. Based on the locality and year of isolation, nine strains were selected for amplification and partial sequencing of the gyrB gene using sets of primers described by Parkinson et al. (2007). Obtained partial DNA sequences showed that all nine strains (GenBank nos. MZ569011, MZ574079, MZ574080, MZ574081, MZ574082, MZ574083, MZ574084, MZ574085, and MZ574086) share 99.86 to 100% identity of gyrB sequence with Xe type strain ICPM:109 as well as 98.71 to 100 % of gyrB sequence identity with Xe strain LMG930 isolated from pepper in The United States. Pathogenicity of all strains was confirmed by spraying young pepper plants (cv. Slonovo uvo) using a handheld sprayer with the bacterial suspension (108 CFU/ml of sterile tap water), in three replicates. Sterile distilled water and reference Xe strain (KFB 1) were used as negative and positive controls, respectively. The inoculated plants were incubated under plastic bags in the greenhouse providing high humidity conditions for 48h. Symptoms were monitored for two weeks after inoculation. Lesions surrounded by a halo appeared on leaves of all inoculated plants within 10 to 15 days after inoculation, while plants inoculated with SDW remained symptomless. Koch's postulates were confirmed by reisolation of the pathogen from necrotic tissue and identity check by PCR using primer set of Koenraadt et al. (2009). The pathogen race was determined according to the reaction of cv. Early Calwonder (ECW) and its isogenic lines (ECW-10R, ECW-20R, ECW-30R) (Stall et al. 2009). Obtained results indicated that all tested strains and reference strain Xe (KFB 1) belong to the pepper race P8. Based on pathogenic, biochemical, and molecular characteristics, the strains isolated from pepper leaves in Montenegro were identified as X. euvesicatoria. Pepper production is particularly significant for small farmers in Montenegro. Favorable climate, use of noncertified seed and lack of crop rotation contributes to the disease occurrence and severity. The disease has probably been around for years but the etiology was not confirmed so far. This is the first report of X. euvesicatoria affecting pepper in this country.

Identification And Detection Of Proteolytic Activity Of Endophite Bacterial Isolated From Avicennia Marina Leaves In Mempawah Mangrove Center

Abstract. Endophytic bacteria are bacteria that live in healthy plant tissue without damaging the plant tissue itself. This study aims to determine the morphological and physiological characteristics, as well as to determine the proteolytic activity of the genus of endophytic bacteria found in Avicennia marina leaves. The research was conducted through the stages of bacterial isolation, morphological characterization, physiological test which includes biochemical factor test and proteolytic activity test, as well as identification. The method used is a systematic sampling method and the data collected were analyzed descriptively. The characteristics of endophytic bacteria obtained have morphological characteristic with rounded colony shapes, flat and wavy edgs, convex elevation, white to yellow colony color, gram positive and negative with cocci and bacillary cell shapes. Physiological tests on the salinity, pH and temperature tests of all isolates produced a positive reaction. The results obtained 6 isolates of endophytic bacteria from Avicennia marina leaves which have similarities with members of the genus Staphylococcus, Bacillus, Pseudomonas and Micrococcus. The isolates obtained did not have proteolytic activity.

Pufferfish (Tetraodon cutcutia) Sampled from a Freshwater River Serves as an Intermediate Reservoir of a Sucrose Nonfermenting Variant of Vibrio cholerae PS-4.

ABSTRACTWe describe the genomic characteristics of Vibrio cholerae strain PS-4 that is unable to ferment sucrose on a thiosulfate citrate bile salt sucrose (TCBS) agar medium. This bacterium was isolated from the skin mucus of a freshwater pufferfish. The genome of strain PS-4 was sequenced to understand the sucrose nonfermenting phenotype. The gene encoding the sucrose-specific phosphotransferase system IIB (sucR) was absent, resulting in the defective sucrose fermenting phenotype. In contrast, genes encoding the glucose-specific transport system IIB (ptsG) and fructose-specific transport system IIB (fruA) showed acid production while growing with respective sugars. The overall genome relatedness indices (OGRI), such as in silico DNA-DNA hybridization (isDDH), average nucleotide identity (ANI), and average amino acid identity (AAI), were above the threshold value, that is, 70% and 95 to 96%, respectively. Phylogenomic analysis based on genome-wide core genes and the nonrecombinant core genes showed that strain PS-4 clustered with Vibrio cholerae ATCC 14035T. Further, genes encoding cholera toxin (ctx), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), toxin-coregulated pilus (tcp), and lipopolysaccharide biosynthesis (rfb) were absent. PS-4 showed hemolytic activity and reacted strongly to the R antibody. Therefore, the Vibrio cholerae from the pufferfish adds a new ecological niche of this bacterium.IMPORTANCE Vibrio cholerae is native of aquatic environments. In general, V. cholerae ferments sucrose on thiosulfate citrate bile salt sucrose (TCBS) agar and produces yellow colonies. V. cholerae strain PS-4 described in this study is a sucrose nonfermenting variant associated with pufferfish skin and does not produce yellow colonies on TCBS agar. Genes encoding sucrose-specific phosphotransferase system IIB (sucR) were absent. The observed phenotype in the distinct metabolic pathway indicates niche-specific adaptive evolution for this bacterium. Our study suggests that the nonfermenting phenotype of V. cholerae strains on TCBS agar may not always be considered for species delineation.

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

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.

Sphingobacterium bovistauri sp. nov., Isolated from the Faeces of Bos Taurus.

A novel bacterium designated WQ 366T was isolated from the faeces of Bos taurus, foraging on the slopes of the Baima Snow Mountain in Yunnan, China. The isolate grew optimally at 30℃ and pH 7.0-8.0 without NaCl. The cells were Gram-stain-negative, aerobic, rod-shaped, non-gliding, catalase-positive, and produced yellow color colonies on Columbia Agar. A polyphasic study was applied to clarify its taxonomic position through 16S rRNA gene and genome sequence analysis, and other extensive biological typing. Phylogenetic analysis revealed that the isolate was affiliated to the genus Sphingobacterium and its 16S rRNA gene sequence was closely related to Sphingobacterium bovisgrunnientis YK2T (97.3%), Sphingobacterium composti T5-12T (96.4%), and Sphingobacterium cavernae 5.0403-2T (96.4%). The calculated whole genome average nucleotide identity (ANI) and the digital DNA-DNA hybridization values between strain WQ 366T and the three related strains were 78.3, 78.6, 73.9 and 21.2, 21.2, 21.0%, respectively. The predominant fatty acids (>10%) were iso-C15:0, iso-C17:0 3-OH, Summed Feature 3 (C16:1 ω7c and/or C16:1 ω6c), and Summed feature 9 (iso-C17:1 ω9c and 10-methyl C16:0). The main polar lipids were PE, GPL, GL, and PL. MK-7 was the major menaquinone. The genome size and the G + C content of WQ 366T was 4.1Mb and 34.6%, respectively. All these results indicated that strain WQ 366T represents a novel species of the Sphingobacterium genus. Therefore, the name Sphingobacterium bovistauri sp. nov. is proposed, and the type strain is WQ 366T (= CCTCC AA 2020029T = KCTC 82395T).

First Report of Xanthomonas campestris pv. campestris Causing Marginal Leaf Necrosis of Arugula (Eruca vesicaria subsp. sativa) in Serbia.

Arugula (Eruca vesicaria subsp. sativa (Miller) Thell., syn. Brassica eruca L.), is an annual cruciferous crop that is increasingly grown for fresh consumption in Serbia. In November 2018, a few detached leaves of cultivated arugula originating from a local producer, showing necrotic lesions, were observed in a fresh vegetable market in Belgrade, Serbia. Information about the disease incidence and severity was not available. Intensity of the observed symptoms was low, but it could be a consequence of the produce quality selection for the market. The leaves developed irregular chlorotic lesions starting from the leaf edge, and tissue within some of them turned dark brown and necrotic (Fig. 1a). From the lesions on different leaves, smooth, bright yellow pigmented, round and opalescent bacterial colonies were isolated on nutrient agar (NA) medium after 72 h of incubation at 26°C. Six bacterial isolates, obtained from three leaf subsamples which induced hypersensitive reaction in tobacco leaves (Nicotiana tabacum L. cv. Samsun), were selected for further studies. On yeast - dextrose - CaCO3 medium, the strains formed characteristic creamy yellow, mucoid, opaque and convex colonies. All isolates were Gram-negative, strictly aerobic, non-fluorescent and catalase positive, did not produce oxidase nor arginine dehydrolase, and did not show pectynolitic activity on potato tuber slices. They hydrolyzed starch, gelatine and esculin, used glucose and sucrose, but not arabinose as a carbon source, and did not reduce nitrates. They grew at 36°C, and tolerated 5% NaCl and 0.02% triphenyl-tetrazolium chloride (Lelliott and Stead, 1987). These growth characteristics were similar as for the reference Xanthomonas campestris pv. campestris (Xcc) strain KFB 105, used in all tests as a positive control (Obradović et al., 2000). The isolates were further characterized by polymerase chain reaction (PCR) using primers DLH120/DLH125, specific for the hrpF gene region of X. campestris according to Berg et al. (2005). Specific DNA fragment of 619 bp was amplified for all tested isolates. Amplification and partial sequencing of the gyrB gene of four isolates was performed using set of primers described by Parkinson et al. (2007). All obtained partial gyrB sequences were identical to each other. According to BLAST analysis (GenBank Acc. Nos. MW508894 - MW508897) they shared 100% of sequence identity with different Xcc strains and 99.5 % with the X.c. pv. raphani pathotype strain, deposited in the NCBI GenBank database. Pathogenicity of the isolates was tested by spraying leaves of 3-week old E. sativa seedlings grown in a commercial potting mix in a greenhouse, with a 24 h-old bacterial culture suspended in sterile distilled water (107 CFU/ml). Xcc strain KFB 105 was used as positive and sterile distilled water as negative control. Inoculated plants were incubated under plastic bags for 48 h and further maintained in a greenhouse at approx. 28°C. On inoculated plants, chlorotic lesions, spreading from the leaf margins, further coalescing into irregular, V-shaped tissue necrosis associated with blackening of veins, developed up to two weeks after inoculation (Fig. 1b, c). The colonies reisolated from symptomatic leaves were identified using PCR, as described above. Based on studied characteristics, all six isolates associated with arugula leaf lesions in Serbia belong to a clonal population. They were identified as X. campestris pv. campestris, the causal agent of black rot, a major disease affecting crucifers, including arugula worldwide (Romero et al., 2008; Rosenthal, et al., 2018). So far, it has been described on Brassica oleracea and B. napus in Serbia (Obradović et al., 2001; Popović et al., 2019). This is the first report of Xcc infecting arugula in this country. The severity of the symptoms developed on artificially inoculated plants indicated significant potential of the pathogen to affect arugula crop in conditions favoring infection. Being a minor crop, accurate information about severity of arugula diseases in Serbia is not available. Lack of crop rotation and close proximity of other Xcc host species on a farm could contribute to further spreading of this problem. Follow up of this arugula disease should reveal the distribution, population structure and genetic diversity of Xcc strains affecting this crop in Serbia.

Antimicrobial Resistance Pattern among Isolated Staphylococcus Aureus Causing Bovine Mastitis in District Khairpur, Sindh, Pakistan

The objective of this study was to determine the incidence of bovine mastitis caused by Staphylococcus aureus and to evaluate antibiotic resistant pattern among isolated S. aureus causing bovine mastitis. A total of 400 cattle milk samples from 3 dairy farms of district Khairpur located in Thehri, Gambat and Khairpur were collected in sterilized bottles. Clinical mastitis was noted based on signs of inflammation in the udder and obvious changes in milk. Subclinical mastitis was evaluated using the California mastitis test (CMT). Bacteria were isolated and identified from milk samples based on cultural, microscopic and biochemical tests. The isolated strains were then tested for their antibiotic sensitivity profile by Kiry-Baur disc diffusion assay. Out of 400 milk samples tested, 255 samples (63.55%) were positive for mastitis as indicated by CMT. S. aureus was isolated from 109 samples (26.25%) as confirmed by yellow colored colonies on Mannitol salt agar, Gram positive cocci in bunches and positive for catalase, coagulase, DNase and beta hemolysis. Area wise prevalence of S. aureus was noted as 31.34%, 28.57% and 21.8% in Khairpur, Thehri and Gambat respectively. S. aureus strains were highly resistant to the antibiotic Meropenem (81.6%) followed by Cefixime (61.4%) Cefotaxime (60.55%), Oxycillin (55%), Erythromycin (54%), Ampicillin (51.3%), Ciprofloxacin (38%), Cefradine (37.6%), Sulphametho-Trim (21.1%), Vancomycin (21.1%), Doxycycline (21.1%) and Amoxicillin-clavulanate (5.5%). This study suggests that clinical and subclinical form of S. aureus associated mastitis is highly prevalent among dairy cattle of district Khairpur and S. aureus has become resistant to most of the antibiotics. Hygiene status of animal is very much essential to control mastitis and to prevent the spread of resistant bacteria to other animals. Keywords: Mastitis; Staphylococcus aureus; Antibiotic Resistance

First Report of Anthracnose Caused by Colletotrichum brasiliense on Violet Passion Fruit in China.

Violet passion fruit (Passiflora edulis Sims) is an important tropical and subtropical perennial evergreen vine with large-scale cultivation in Guangxi, China. Between May and September 2020, anthracnose symptoms occurred on passion fruit (cultivar Tainong No. 1) in Xingye county (22°77'13″N, 110°07'80″E) in Guangxi province, China. The disease incidence varied from 25 to 60% in different orchards. Initial symptoms on young fruits appeared as multiple tiny water-soaked, oval to irregular pale greenish spots. As the disease progressed, the lesions became medium brown, with sunken cavities. Under humid conditions, acervuli containing masses of conidia and dark setae were found on the lesions. The affected fruits became shriveled. Tissue pieces (5 × 5 mm) were cut out from infected fruits, surface sterilized in 75% ethanol for 15 s and 0.1% HgCl2 for 2 min, washed three times with sterile water, placed onto potato dextrose agar (PDA), and incubated at 28 °C for three days. Of the 29 Colletotrichum isolates obtained , the isolate B13 was selected for morphological characterization. B13 was purified by single spore isolation and incubated on PDA at 25°C under continuous fluorescent light irradiation, producing white to pale yellow colonies with dense aerial mycelia. The reverse side of the colony was pale yellowish to olive. Conidia were hyaline, unicellular, straight, cylindrical, with both ends slightly round or one end round and the other slightly pointed, measuring 10.5 to 18.8 (average 16.4) × 5.4 to 7.2 (average 6.3) µm (n = 50). Appressoria were light brown to dark black, smooth-walled, lobed, often with a roundish outline, sometimes also triangular, 7.2 to 10.9 (average 9.1) × 6.8 to 9.2 (average 8.2) µm (n = 50). Morphological characteristics of the isolate matched those of Colletotrichum brasiliense (Damm et al. 2012). The internal transcribed spacers (ITS), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-tubulin (TUB2) genes of strain B13 were sequenced using the method and primers of Damm et al. (2012). Sequences of the amplified DNA regions were submitted to GenBank (ITS: MW198820; ACT: MW266083; GAPDH: MW266084; and TUB2: MW266085). A concatenated maximum likelihood phylogenetic tree was built using MEGA 7.0.21 in which B13 clustered with C. brasiliense and clearly separated from other Colletotrichum spp. Pathogenicity of B13 was assayed using one-year-old plants of violet passion fruit cultivar 'Tainong No. 1'. Conidial suspensions were prepared from 7-day-old cultures grown on PDA at 28°C Sterile distilled water was used to dislodge conidia from the culture dish and the conidial concentration was adjusted to 1 × 106 spores mL-1 using a haemocytometer. Fruits were rinsed with sterilized water and wounded with a sterile needle at three locations. Three fruits were inoculated by spraying with 20 mL of the conidial suspension. Control fruits were sprayed with distilled water. Fruits were then covered with plastic bags to maintain high relative humidity . After 9 days, all inoculated fruits developed brown spots with sunken cavities, resembling symptoms observed in the field, and controls remained symptomless. Fungal cultures with phenotypic features similar to C. brasiliense were re-isolated from the symptomatic fruits, verifying C. brasiliense as the causal agent of the disease based on Koch's postulates. C. boninense, C. gloeosporioides, C.queenslandicum, C. brevisporum, and C. karstii were reported as causal agents of anthracnose on passion fruit (Júnior et al.2010; Power et al. 2010; James et al.2014; Du et al.2017; Ran et al.2020). To the best of our knowledge, this is the first report of C. brasiliense causing anthracnose on passion fruit in China.

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

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

Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal

Cholera is a gastrointestinal disease caused by pathogenic strain of Vibrio cholerae, the disease clinically manifested by rice-water diarrhea, nausea and vomiting. This study aimed to study the incidence of Vibrio species and employ simple method for rapid detection of Vibrio cholerae from water samples of Sunsari, Nepal. Identification of V. cholerae through biochemical tests requires extensive labor and costs. In resource limited laboratories, isolation and identification of V. cholerae often becomes difficult. Therefore, this study also aimed for selecting scope of this methodology as a scientific outcome for rapid isolation and identification of Vibrio cholerae. A total of 100 water samples were collected from Sunsari district in which 25 samples were collected from sewage, 25 from pond, 25 from tap and 25 from tube well. The samples of collected water were sent to the microbiology laboratory of Central Campus of Technology maintained in ice cold box and were enriched in Alkaline Peptone water and selectively isolated from TCBS agar and NA agar without NaCl. Pathogens were isolated and identified by conventional microbiological techniques. Out of 100 water samples collected, sucrose fermenting Vibrio species were isolated only from 16 water samples. Further the selective isolation of V. cholerae from nutrient agar without NaCl isolated 6 isolates from sewage samples and 3 isolates from pond samples. The distribution of Vibrio cholera in the water sample was found to be 9%, distribution of V. alginolyticus was found to be 4% and distribution of V. fluvialis was found to be 3%. In this study, non-sucrose fermenting Vibrio species were not isolated from the water samples. However, sucrose fermenting Vibrio species was obtained with yellow pigmentation in TCBS agar medium. The yellow pigmented colonies of Vibrio isolates recovered from TCBS and even from Nutrient Agar devoid of sodium chloride provided sufficient evidence of V. cholerae after series of other biochemical tests. This study concludes that yellow colonies (sucrose-fermenting) of Vibrio from TCBS agar medium that can grow on nutrient agar without added NaCl and which exhibit a positive oxidase reaction can be confidently identified as presumptive V. cholerae. In resource-constrained environments, this simple method can reduce the labor cost, chemicals and time-consuming procedure of performing multiple biochemical and molecular assays for identification.

Xanthomonas hydrangeae sp. nov., a novel plant pathogen isolated from Hydrangea arborescens.

This paper describes a novel species isolated in 2011 and 2012 from nursery-grown Hydrangea arborescens cultivars in Flanders, Belgium. After 4 days at 28 °C, the strains yielded yellow, round, convex and mucoid colonies. Pathogenicity of the strains was confirmed on its isolation host, as well as on Hydrangea quercifolia. Analysis using MALDI-TOF MS identified the Hydrangea strains as belonging to the genus Xanthomonas but excluded them from the species Xanthomonas hortorum. A phylogenetic tree based on gyrB confirmed the close relation to X. hortorum. Three fatty acids were dominant in the Hydrangea isolates: anteiso-C15 : 0, iso-C15 : 0 and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c). Unlike X. hortorum pathovars, the Hydrangea strains were unable to grow in the presence of lithium chloride and could only weakly utilize d-fructose-6-PO4 and glucuronamide. Phylogenetic characterization based on multilocus sequence analysis and phylogenomic characterization revealed that the strains are close to, yet distinct from, X. hortorum. The genome sequences of the strains had average nucleotide identity values ranging from 94.35-95.19 % and in silico DNA-DNA hybridization values ranging from 55.70 to 59.40 % to genomes of the X. hortorum pathovars. A genomics-based loop-mediated isothermal amplification assay was developed which was specific to the Hydrangea strains for its early detection. A novel species, Xanthomonas hydrangeae sp. nov., is proposed with strain LMG 31884T (=CCOS 1956T) as the type strain.

Cerebral abscesses in Marfan’s disease

A 43-years-old woman was hospitalized with headaches progressing over the 10 preceding days and an epileptic seizure. The patient had a Marfan disease which required two cardiac surgeries - the last one in 2014 with the installation of an aortic tube and a mechanical aortic valve. A gadolinium-enhanced magnetic resonance image of the brain was performed and revealed numerous lesions, some with ring enhancement, substantial edema but without mass effect (Figure 1). Additional tests were carried out including blood cultures, transthoracic and trans-oesphageal echocardiogram, Coxiella burnetii, Bartonella spp. and Mycoplasma pneumonia serologies that remained all negative. After 48 h incubation of CT-guided brain abscess aspiration, small yellow colonies appear on chocolate blood agar and Gram staining shows small Gram-negative bacilli (Figures 2,3). Identification realized by MALDI-TOF mass spectrometry revealed Aggregatibacter aphrophilus. Blood cultures remained sterile.

P.0734 Pisa syndrome with lithium treatment: case report and literature review

A 23-year-old male patient who was a follow-up case of neurosurgery presented to our emergency department with a history of high-grade fever and clinical features of meningitis for 1 week. The cerebrospinal fluid (CSF) was sent to our laboratory for culture. The culture demonstrated growth of 1-2 mm in diameter light yellow coloured colonies of Gram-negative bacilli on chocolate and blood agar. There was no growth on MacConkey agar. The bacterium was multidrug resistant. Based upon the growth characteristics, bio-chemical reactions, drug susceptibility pattern and identification by Vitek 2 system the isolate was identified as Elizabethkingia meningoseptica. Patient was treated with injection piperacillin-tazobactam, injection vancomycin and cotrimoxazole tablets for 21 days along with intrathecal injection of tigecycline and finally, patient improved clinically and the CSF cultures became sterile. The presence in hospital environment along with multidrug resistance makes E. meningoseptica a successful emerging nosocomial pathogen.

Morphological and molecular characterization of Burkholderia glumae causing panicle blight of paddy

Bacterial panicle blight (BPB) is one of the emerging diseases of paddy in the Tarai region of Uttarakhand. The present investigation was conducted to isolate and identify the cause of BPB, to detect the pathogen longevity in in-vitro stored paddy seeds and glass house conditions and to identify the most susceptible stage for pathogen infection. 42 rice panicle samples from different paddy growing regions of the Tarai areas were analyzed, and the bacterium was isolated from different parts of paddy seeds. The isolated bacterial strains were characterized culturally in semi-selective media (S-PG), morphologically (SEM) and molecularly (16S rRNA). A total of 16 samples produced yellow colored colonies on King's B medium and 9 samples produced brisk yellow colored colonies on the S-PG agar suggesting them to be of B. glumae. However with PCR reactions we found that 2 strains out of 9 were of B. glumae and the causal agent for the other sample remains unknown. Recovery of bacterium from infected stored seeds was also studied at monthly intervals from harvest to next growing season and maximum recovery was observed from the seeds just after harvest (91%) which although decreased with the storage period but the bacterium did not get completely eliminated during the course of storage. On exploring the different growth stages for most susceptible stage of infection, boot leaf and panicle initiation stages were found to be equally susceptible with 93.33% disease incidence. These findings suggest that B. glumae is distributed generally in the paddy seed and can also survive in it for a longer duration thereby acting as a source of inoculums for the next cropping season. These findings will help to understand the mode of survival of bacteria in plants and its longevity in seeds.

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

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.

First Report of Strawberry Crown Rot Caused by Xanthomonas fragariae in China.

Strawberry (Fragaria × ananassa Duch.) is a kind of fruit with great economic importance and widely cultivated in the world. From 2019 to 2020, a serious crown rot disease was sporadically observed in several strawberry cultivars including 'Zhang Ji', 'Hong Yan' and 'Yue Xiu' in Shanghai, China. Initially, water-soaked rot appeared in inner tissue of strawberry crown, then progressed into browning and hollowing symptoms accompanied with yellow discolorations of young leaves. To isolate and identify the causal agent, small pieces of tissue taken from ten diseased crowns were sterilized by 70% alcohol. The cut-up pieces were macerated and serially diluted. The dilutions were placed on nutrient agar (NA) medium. After incubation at 25°C for 4-5 days, the yellow bacterial colonies were tiny and were streaked on NA plate for purification. The colonies were yellow, mucoid, smooth-margined, and five independent representative colonies were used for further confirmation. To confirm the species identity of the bacterial, genomic DNA was extracted from the five representative isolates, and 16S rRNA gene was amplified and sequenced using universal primers 27F/1492R. The 16S rRNA sequence was deposited in GenBank (MW725235) and showed 99% nucleotide similarity with Xanthomonas fragariae strain LMG 708 (NR_026318). The isolate's identity was further confirmed by X. fragariae-specific primers XF9/XF12 (Roberts et al. 1996). All five isolates could be detected by XF9/XF12 primer. To confirm Koch's postulates, five healthy strawberry plants were placed in 1000 ml glass beakers by submerging the cutting wound in 50 ml the bacterial suspension of 108 CFU/ml. Five additional strawberry plants treated with sterilized water served as a control. The beakers containing inoculated plants were sealed with plastic film at 25°C. Water-soaked rot appeared on internal tissue of crown similar to those observed in the field within 10-12 days after inoculation, while the control samples remained healthy. The bacteria were re-isolated from rot of inoculated crowns, and confirmed by X. fragariae-specific primers XF9/XF12. X. fragariae has been reported to cause angular leaf spot on strawberry in China (Wang et al. 2017; Wu et al., 2020). It's also found that X. fragariae could systematically infect crown tissue (Milholland et al. 1996; Mahuku and Goodwin, 1997). To our knowledge, this is the first report of X. fragariae causing strawberry crown rot in China. This report increased our understanding of X. fragariae, and showed that the spread of this disease might seriously threaten the development of strawberry industry in the future.

Genomic molecular signatures determined characterization of Mycolicibacterium gossypii sp. nov., a fast-growing mycobacterial species isolated from cotton field soil.

A Gram-positive, acid-fast and rapidly growing rod, designated S2-37T, that could form yellowish colonies was isolated from one soil sample collected from cotton cropping field located in the Xinjiang region of China. Genomic analyses indicated that strain S2-37T harbored T7SS secretion system and was very likely able to produce mycolic acid, which were typical features of pathogenetic mycobacterial species. 16S rRNA-directed phylogenetic analysis referred that strain S2-37T was closely related to bacterial species belonging to the genus Mycolicibacterium, which was further confirmed by pan-genome phylogenetic analysis. Digital DNA-DNA hybridization and the average nucleotide identity presented that strain S2-37T displayed the highest values of 39.1% (35.7-42.6%) and 81.28% with M. litorale CGMCC 4.5724T, respectively. And characterization of conserved molecular signatures further supported the taxonomic position of strain S2-37T belonging to the genus Mycolicibacterium. The main fatty acids were identified as C16:0, C18:0, C20:3ω3 and C22:6ω3. In addition, polar lipids profile was mainly composed of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. Phylogenetic analyses, distinct fatty aids and antimicrobial resistance profiles indicated that strain S2-37T represented genetically and phenotypically distinct from its closest phylogenetic neighbour, M. litorale CGMCC 4.5724T. Here, we propose a novel species of the genus Mycolicibacterium: Mycolicibacterium gossypii sp. nov. with the type strain S2-37T (= JCM 34327T = CGMCC 1.18817T).

Effect of Biofloc product‐Rapid BFT TM vs. clear water system in improving the water quality and growth performances of Pacific Whiteleg shrimp, P. vannamei , cultured in indoor aquaculture system

Biofloc technology was known to improve the shrimp growth performance and water quality in several ways. The application of bacterial booster was proven to accelerate the development of biofloc in the culture treatments as well as help improve the water quality, shrimp growth performance and yield production. Due to its importance, a study was conducted to identify the effect of biofloc application using Rapid BFTTM microbial booster formulation versus clear water system to improve shrimp growth performance and water quality condition. Study was conducted for 80 days with every 7 days of interval of data collection. Overall, biofloc treatments were identified successfully in improving the shrimp growth performance and also effectively reduced the nutrient concentrations of total ammonia nitrogen (TAN) from the biofloc culture treatments until the end of culture periods. Nitrate (NO3−) from biofloc treatments was also identified in lower concentration as compared to control of clear water system. The average daily growth (ADG) was at 0.12g much better from biofloc treatments as compared to control with ADG 0.08g of clear water system. The specific growth rate (SGR) was 5.47%/day in biofloc and 2.82%/day in control treatments which identified higher and much better from biofloc about 2.65%/day differences between biofloc and clear water. Meanwhile, the final body weight achieved was much higher from biofloc about 9.55 grams compared with control about 7.06 grams from the same day of culture, DOC80 which recorded at the end of cultured period. The first sampling was started at DOC7 where the average initial weight of shrimp in biofloc treatment was 0.117 grams and 0.077 grams identified in control. The highest biomass achieved from biofloc treatments also much better around 11.0 kg compared with control that produced 7.1 kg as the higher biomass produced. There was only yellow and green colony of Vibrio identified from the TCBS culture and no occurrence of luminescent colony from harmful Vibrio harveyi detected. Therefore, it can be concluded that biofloc technology of Rapid BFTTM help improves the water quality of ammonia nitrogen level in water columns and inhibits pathogenic Vibrio to the culture species as well as help improving the shrimp growth performances in more effective way.

Streptomonospora litoralis sp. nov., a halophilic thiopeptides producer isolated from sand collected at Cuxhaven beach

Strain M2T was isolated from the beach of Cuxhaven, Wadden Sea, Germany, in course of a program to attain new producers of bioactive natural products. Strain M2T produces litoralimycin and sulfomycin-type thiopeptides. Bioinformatic analysis revealed a potential biosynthetic gene cluster encoding for the M2T thiopeptides. The strain is Gram-stain-positive, rod shaped, non-motile, spore forming, showing a yellow colony color and forms extensively branched substrate mycelium and aerial hyphae. Inferred from the 16S rRNA gene phylogeny strain M2T affiliates with the genus Streptomonospora. It shows 96.6% 16S rRNA gene sequence similarity to the type species Streptomonospora salina DSM 44593 T and forms a distinct branch with Streptomonospora sediminis DSM 45723 T with 97.0% 16S rRNA gene sequence similarity. Genome-based phylogenetic analysis revealed that M2T is closely related to Streptomonospora alba YIM 90003 T with a digital DNA-DNA hybridisation (dDDH) value of 26.6%. The predominant menaquinones of M2T are MK-10(H6), MK-10(H8), and MK-11(H6) (> 10%). Major cellular fatty acids are iso-C16:0, anteiso C17:0 and C18:0 10-methyl. The polar lipid profile consisted of diphosphatidylglycerol phosphatidyl glycerol, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, three glycolipids, two unknown phospholipids, and two unknown lipids. The genome size of type strain M2T is 5,878,427 bp with 72.1 mol % G + C content. Based on the results obtained from phylogenetic and chemotaxonomic studies, strain M2T (= DSM 106425 T = NCCB 100650 T) is considered to represent a novel species within the genus Streptomonospora for which the name Streptomonospora litoralis sp. nov. is proposed.

First Report of Xanthomonas fragariae strain YL19 causing crown infection pockets in strawberry in Liaoning Province, China.

Strawberry (Fragaria × ananassa Duch.) is an important fruit crop in China. Typical crown infection pockets symptoms were observed on the infected strawberry in Liaoning province, China (121°60'E, 38°90'N) in the autumn of 2017. The disease incidence was estimated to be around 5 to 10 %, but could reach 30 to 40 % in some heavily affected plastic tunnels. The infected plants early displayed water-soaked symptoms on the abaxial leaf surface and subsequently developed reddish-brown shaped stripes and coalesced lesions on the adaxial leaf surface around the main veins (Fig. 1-A, 1-B). Several variable-size (0.3-0.8 mm in diameter) pockets were observed inside the crown tissues after dissection (Fig. 1-C). The diseased plants rarely reached fruiting and were easily broken between the crown tissue and the stem, and would eventually die. To identify the causal agent of this disease, the several surface-disinfested infected main veins and crown tissues were individually ground in sterile water and plated on sucrose peptone agar(SPA) medium (Hayward 1960) with 10-fold serial dilutions and incubated at 25℃. A number of yellow colonies grew on the medium at the 10-4 dilution 7 days after plating (Fig. 1-D) in all specimens. The colonies were aerobic, yellow, viscous, smooth, and gram-negative, which is a typical characteristic of Xanthomonas. To confirm identity of the causal bacteria, 18 colonies selected randomly were subjected to polymerase chain reactions (PCR) for the amplification of the cpn60 (Sahin et al. 2010), gyrB, rpoD, and fyuA (C Manceau et al. 2011), respectively. The results showed that the 18 colonies are identical. The cpn60, gyrB, rpoD, and fyuA sequences of this isolate were deposited in GenBank with accession numbers MT513132.1, MW233896, MW233897, and MW233895, respectively. BLAST searches with sequences of this isolate cpn60, gyrB, rpoD, and fyuA revealed 97.7%, 96.4%, 97.8%, and 97.3% similarity with the corresponding sequences of X. fragariae strain NBC2815 (LT853880.1), respectively. The resulting concatenated data set of cpn60-gyrB-rpoD-fyuA was used to build a Multilocus Sequence Analysis (MLSA) by maximum likelihood criteria (Fig. 2). The cpn60-gyrB-rpoD-fyuA sequences of the isolate from Liaoning clustered in the clade containing the type strain of X. fragariae NBC2815, indicating that it belongs to X. fragariae. Thus, the bacterial strain from Liaoning was designated as X. fragariae strain YL19. To fulfill Koch's postulates, the base of leaf petioles of disease-free strawberry plants were syringe-infiltrated inoculated with bacterial suspension (2×108 CFU) prepared from colonies of X. fragariae YL19 washed from SPA plates. The inoculated and control (treated with sterile water) were placed in a chamber (25/20℃day/night,≥90% relative humidity(RH), 12/12 h photoperiod) for three months. After one month, water-soaked symptoms were observed in the crown tissues of all X. fragariae YL19-inoculated plants. Two months after inoculation, a significant crown pocket similarly to initial symptoms observed in the field was developed on all inoculated plants. No symptoms were observed in the control plants. The bacteria were re-isolated from the symptomatic leaves, petioles and crowns, and confirmed as X. fragariae YL19 by the above mentioned morphological and molecular analyses. Pathogenicity tests were conducted three times and the same results were obtained. It was reported that X. fragariae usually causes angular leaf spot, a serious bacterial disease in many strawberry production regions worldwide. The typical symptoms of angular leaf spot caused by X. fragariae include reddish-brown, irregular spots on the upper leaf surface, water-soaked lesions developed along leaf veins. Although angular leaf spot caused by X. fragariae has been reported in Tianjin and Taiwan province, China (Wang et al. 2017; Wu et al. 2020), there is no report about the symptoms that infection pockets on crowns caused by X. fragariae strain YL19 as described above. This result indicated that YL19 is different from the other two X. fragariae strains reported in China or the disease caused by YL19 could be a severe case of angular leaf spot and vascular decline or collapse in strawberry (Bradbury, 1977). . To the best of our knowledge, these results showed a previously unreported new strain YL19 of X. fragariae is the causal agent of crown infecton pocket in strawberry in China, it may lead to serious losses to the local strawberry industry. This report will assist in developing management measures for this disease promptly.