Pale Yellow Colonies Research Articles

Whole genome sequencing and characterization of Pantoea agglomerans DBM 3797, endophyte, isolated from fresh hop (Humulus lupulus L.).

This paper brings new information about the genome and phenotypic characteristics of Pantoea agglomerans strain DBM 3797, isolated from fresh Czech hop (Humulus lupulus) in the Saaz hop-growing region. Although P. agglomerans strains are frequently isolated from different materials, there are not usually thoroughly characterized even if they have versatile metabolism and those isolated from plants may have a considerable potential for application in agriculture as a support culture for plant growth. P. agglomerans DBM 3797 was cultured under aerobic and anaerobic conditions, its metabolites were analyzed by HPLC and it was tested for plant growth promotion abilities, such as phosphate solubilization, siderophore and indol-3-acetic acid productions. In addition, genomic DNA was extracted, sequenced and de novo assembly was performed. Further, genome annotation, pan-genome analysis and selected genome analyses, such as CRISPR arrays detection, antibiotic resistance and secondary metabolite genes identification were carried out. The typical appearance characteristics of the strain include the formation of symplasmata in submerged liquid culture and the formation of pale yellow colonies on agar. The genetic information of the strain (in total 4.8 Mb) is divided between a chromosome and two plasmids. The strain lacks any CRISPR-Cas system but is equipped with four restriction-modification systems. The phenotypic analysis focused on growth under both aerobic and anaerobic conditions, as well as traits associated with plant growth promotion. At both levels (genomic and phenotypic), the production of siderophores, indoleacetic acid-derived growth promoters, gluconic acid, and enzyme activities related to the degradation of complex organic compounds were found. Extracellular gluconic acid production under aerobic conditions (up to 8 g/l) is probably the result of glucose oxidation by the membrane-bound pyrroloquinoline quinone-dependent enzyme glucose dehydrogenase. The strain has a number of properties potentially beneficial to the hop plant and its closest relatives include the strains also isolated from the aerial parts of plants, yet its safety profile needs to be addressed in follow-up research.

Jatrophihabitans cynanchi sp. nov., isolated from rhizosphere soil of Cynanchum wilfordii.

A novel actinobacterial strain, SB3-54T was isolated from rhizosphere soil of Cynanchum wilfodill, Jaecheon, Chungcheongbuk-do, Republic of Korea. Cells of strain SB3-54T were Gram-stain-positive, aerobic, rod-shaped, and flagellated which formed pale yellow colonies on Reasoner's 2A (R2A) agar. Growth occurred at 15-30°C (optimum 25°C), pH 5-8 (optimum pH 7), and 0-2.5% NaCl (optimum 0%). Phylogenetic and phylogenomic analyses showed that strain SB3-54T formed a separate lineage in the genus Jatrophihabitans with Jatrophihabitans telluris N237T. Strain SB3-54T was positive for catalase activity. Genomic analysis showed that SB3-54T has plant-beneficial function contributing (referred to as PBFC) genes such as root colonization and plant protection from oxidative stress. Furthermore, genome of SB3-54T contained gene clusters related to cytokinin biosynthesis, auxin response, tryptophan biosynthesis, siderophore biosynthesis and bacterial toxin-antitoxin systems. Strain SB3-54T contained iso-C16:0 as the major fatty acid and MK-9(H4) and MK-9(H6) as the predominant quinones. The organism had meso-diaminopimelic acid as the diagnostic diamino acid in the peptidoglycan. The major polar lipids were diphosphatidylglycerol, phosphatidylinositol polymannosides, two unidentified aminoglycophospholipids and three unidentified phospholipids. Based on phylogenetic, physiological and chemotaxonomic characteristics, strain SB3-54T represents a novel species of the genus Jatrophihabitans. The type strain is SB3-54T (= KCTC 49134T = NBRC 114108T).

Lysobacter arvi sp. nov. Isolated from Farmland Soil.

A bacterial strain designated as UC was isolated from farmland soil. Strain UCT formed a pale yellow colony on nutrient agar. Cell morphology revealed it as the rod-shaped bacterium that stained Gram-negative. The 16S rRNA gene sequence analysis identified strain UCT as a member of the genus Lysobacter that showed high identity with L. soli DCY21T (99.5%), L. panacisoli CJ29T (98.7%), and L. tabacisoli C8-1T (97.9%). It formed a distinct cluster with these strains in the neighbor-joining phylogenetic tree. A similar tree topology was observed in TYGS-based phylogenomic analysis. However, genome sequence analyses of strain UCT showed 87.7% average nucleotide identity and 34.7% digital DNA-DNA hybridization similarity with the phylogenetically closest species, L. soli DCY21T. The similarity was much less with other closely related strains of the genus Lysobacter. The G + C content of strain UCT was 68.1%. Major cellular fatty acids observed were C14:0 iso (13.4%), C15:0 iso (13.6%), and C15:0 anteiso (14.8%). Quinone Q-8 was the major respiratory ubiquinone. Predominant polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, and phosphatidylglycerol. Production of xanthomonadin pigment was observed. Based on phenotypic differences and phylogenomic analysis, strain UCT represents a novel species of the genus Lysobacter, for which the name Lysobacter arvi is proposed. The type strain of the novel species is UCT (= KCTC 92613T = JCM 23757T = MTCC 12824T).

Kosakonia cowanii, a new bacterial pathogen affecting foxtail millet (Setaria italica[L.]P. Beauv.) in China

Foxtail millet (Setaria italica [L.] P. Beauv.) is an important cereal worldwide. From 2021 to 2022, stalk rot disease of foxtail millet was identified in Shanxi province, northern China, with an 8% and 2% field incidence rate in Xinzhou (2 different locations), respectively. It caused necrosis, decay, stem lodging, and sometimes death. This study aimed to identify the causal agent of the disease through morphophysiological and molecular identification of the isolates. Stalk rot specimens were collected in Xinzhou, from foxtail millet plants exhibiting typical symptoms, and the pathogen was isolated with dilution plating. It was cultured at 28 °C for 48 h on nutrient agar, revealing circular, convex, and pale-yellow colonies, with a smooth surface and an entire edge. Scanning electron microscopy showed that the pathogen is rod shaped, round ended and has an uneven surface ranging from 0.5 to 0.7 μm in diameter and 1.2–2.7 μm in length. It is a motile gram-negative facultative anaerobic bacterium that can reduce nitrate and synthesize catalase but cannot hydrolyze starch. It also shows a negative reaction in the methyl red test and optimum growth at 37 °C. The pathogenicity test was performed on foxtail millet variety ‘Jingu 21’ stem to confirm Koch's postulates. The biochemical tests were done in the Biolog Gen III MicroPlate, revealing 21 positive chemical sensitivity tests, except those for minocycline and sodium bromate. Furthermore, among 71 carbon sources, the pathogen utilized 50 as the sole carbon source, including sucrose, d-maltose, α-d-lactose, d-galactose, D-sorbitol, D-mannitol, glycerol, and inositol. Finally, molecular characterization of the pathogen using 16S rRNA and rpoB gene sequencing and subsequent phylogenetic analysis identified the strain as Kosakonia cowanii. This study is the first to report K. cowanii as a stalk rot-causing pathogen in foxtail millet.

First Report of Nalanthamala psidii causing wilt disease of guava in Guangdong, China.

Guava (Psidium guajava L.) is a tropical fruit with great economic value. Guangdong is one of the most important guava production areas. In November 2019, guava wilt disease (GWD) was observed in a 10.6 HA commercial orchard in NanSha district, Guangzhou, Guangdong (22°37'37.626" N, 113°35'56.089" E). Disease incidence was up to 35%. Initially, leaves on the top of some branches became purple or yellow interveinal chlorosis, later dry. Infection severely became systemic developing vascular discoloration of stem, black root rot, eventually entire trees wilted and died. The root tissues were cut into 5-mm2 pieces and surface disinfected with 70% ethanol for 30 sec, 3 % sodium hypochlorite for 4 min, rinsed by the sterile water, then plated onto potato dextrose agar and incubated for 5 days at 25°C. A total of 8 monoconidial isolates with identical colony morphology were obtained. All formed cottony, whitish to pale yellow colonies. Conidiophores were dimorphic, penicillate and acremonium-like. Penicillate conidiophores gave rise to ovoidal, one-celled conidia (4.15 to 6.55×2.28 to 4.61 μm) (n=100) with truncated ends. Cylindrical or fusiform conidia (7.02 to 15.57×2.01 to 5.30 μm) (n=100) arose in long chains on acremonium-like conidiophores. Morphological characteristics of the isolates were consistent with those of Nalanthamala psidii (syn. Myxosporium psidii) reported by Schroers (2005). The rDNA internal transcribed spacer (ITS) and partial nuclear large-subunit ribosomal DNA (LSU) of two representative isolates (GDNS02 and GDNS08) were amplified using the primers pairs ITS4/ITS5 (White et al. 1990) and V9G/LR5 (de Hoog and Gerrits van den. 1998), respectively. The obtained sequences were deposited in GenBank under the accession nos. OM278372 to 73 (ITS) and OM278377 to 78 (LSU). BLASTn analysis showed 99.81% and 100% identities with the reported sequences of N. psidii CBS 116952 (AY864836) and CBS 110507 (AY554243). Maximum likelihood analyses of combined ITS and LSU sequences indicated that these two isolates being clustered with N. psidii strains. Pathogenicity tests were performed twice using healthy seedlings (60-70 cm height, cv. pearl). Each stem of five seedlings was wounded using a 5-mm sterile cork borer, and 5-day-old mycelium plugs of isolate GDNS08 were inoculated into the holes (25-cm above the soil line) and covered with Parafilm, sterile PDA plugs were placed into the wounds of additional 5 control seedlings. All plants were kept in a greenhouse (25℃, 80% relative humidity, 16/8-h day/night). After 3 months, all inoculated plants developed purple leaf, defoliation and wilt symptoms resembling those observed in the orchards, while the controls remained asymptomatic. Nalanthamala psidii was reisolated from the roots tissue of the inoculated plants, identity was confirmed by morphological characteristics and ITS sequence analyses as described above, but not from the controls, fulfilling Koch's postulates. Nalanthamala psidii has been previously reported as the causal agent of guava wilt in Taiwan, Philippines, South Africa and Bangladesh (Hsieh et al. 1976; Opina 1995; Schoeman et al. 1997; Alam et al. 2019). To our knowledge, this is the first report of N. psidii causing guava wilt in Guangdong, China. The outbreak of GWD in South Africa in the 1980s resulted in devastating losses to guava industry (Schoeman et al. 1997). Further research is needed to develop the integrated management to constrain this disease from spreading.

Arthrobacter caoxuetaonis sp. nov., Arthrobacter zhangbolii sp. nov. and Arthrobacter gengyunqii sp. nov., isolated from Marmota himalayana faeces from Qinghai-Tibet Plateau.

Six aerobic or facultative anaerobic, motile, Gram-stain-positive, catalase-positive and oxidase-negative strains (zg-Y453T, zg-Y324, zg-Y462T, zg-Y411, zg-Y809T and zg-Y786) were isolated from different faecal samples of Marmota himalayana from the Qinghai-Tibet Plateau. Pale yellow, round, raised and moist colonies appeared 48 h after incubation at 28 °C on brain-heart infusion plates supplemented with 5 % defibrinated sheep blood. According to the 16S rRNA gene sequence alignment, two strain pairs (zg-Y453T/zg-Y324 and zg-Y462T/zg-Y411) shared the highest similarities to Arthrobacter luteolus (99.5 and 99.2 %), and the other one (zg-Y809T/zg-Y786) to Arthrobacter citreus (99.5 %). Results of phylogenetic analysis based on the 16S rRNA gene and genome sequences showed that these six strains represented three separate species within the genus Arthrobacter. The average nucleotide identity and digital DNA-DNA hybridization values between the three novel type strains (zg-Y453T/zg-Y462T/zg-Y809T) and other known species in this genus were all below respective thresholds (70.2-81.5/19.6-24.2 %, 70.6-81.8/19.8-25.0 %, and 70.4-88.2/19.9-35.3 %). Although phylogenetically related, there were obvious chemotaxonomic and phenotypic differences: strain pair zg-Y462T/zg-Y411 had anteiso-C15 : 0 as the only major fatty acid; the three novel species had different dominant quinones, MK-8(H2) in strains zg-Y462T/zg-Y809T (74.8/81.1 %) and MK-8(H2)/MK-9(H2) (43.1/53.0 %) in zg-Y453T; similarly, the ability to reduce nitrate in strains zg-Y453T and zg-Y462T could differentiate them from zg-Y809T. All strains had diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol, but differed slightly in the types of unidentified glycolipids, phospholipids and lipids. Based on the results of these polyphasic taxonomic analyses, three novel species within the genus Arthrobacter are proposed, namely Arthrobacter caoxuetaonis sp. nov. (type strain, zg-Y453T=GDMCC 1.2809T=JCM 35173T), Arthrobacter zhangbolii sp. nov. (type strain, zg-Y462T=GDMCC 1.2880T=JCM 35170T) and Arthrobacter gengyunqii sp. nov. (type strain, zg-Y809T=GDMCC 1.2808T=JCM 35168T).

Black rot disease incited by Indian race 1 of Xanthomonas campestris pv. campestris in Brassica juncea L. cv. Pusa Bold in India.

Mustard (Brassica juncea L.) is an important oil seed crop in the Brassicaceae family. It is widely cultivated in India for its edible leaves, oil and medicinal properties. In January 2022, we noticed necrotic symptoms typical black rot disease on Brassica juncea (L.) cv. Pusa Bold grown in Indian Agricultural Research Institute, India. Initially, chlorotic lesions emerged on the leaf margin, which progressed to angular V-shaped necrotic lesions and blackened veins. Disease progression became a necrotic appearance in the leaf results browning and papery leaf texture appeared. The suspected causal agent was isolated from three different diseased plants of Pusa Bold on nutrient sucrose agar medium that formed pale yellow, mucoid, and fluidal colonies. Three representative isolates originated from three different plants were sub-cultured on YGCA medium. These isolates are Gram-negative, oxidase negative, KOH positive, nonfluorescent on King's Medium B agar, and positive for starch hydrolysis test (Schaad and White 1974). The 16S ribosomal RNA gene and avirulence genes - AvrBs1 and AvrGf1 were amplified and sequenced in these three isolates with other Xanthomonas campestris pv. campestris (Xcc) isolates. The DNA sequence analysis revealed that these isolates are within the species of X. campestris. The race 1 specific marker namely xcc-b100_4389 was used to characterized the race by detection of 1090bp fragment respectively from gDNA of Xcc isolates (Rubel et al., 2017). The pathogenicity of these isolates was tested twice on youngest leaves of 30-day-old plants of Pusa Bold to convey Koch postulates. Inoculum of three isolates were prepared in nutrient broth at 28°C for 48-h. The pathogenicity test was conducted by small scissors dipped in a bacterial suspension (~ 108 cfu/ml) to cut leaf near margins at 10 points per leaf and the three youngest leaves per plant with three replications. The number of infected points per leaf and the severity of symptoms were assessed 15 and 30 days after inoculation (Singh et al., 2011; 2016). The chlorotic lesions with V-shaped symptoms were appeared on all inoculated plants after 15 and 30 dpi (days post-inoculation). The bacteria were reisolated from inoculated plants and has the same identity as original isolates by using 16S rRNA, avr genes and race 1 specific marker PCR, thereby confirming Koch's postulates. The bacterial inoculation was repeated and the same symptoms appear. Most of the crucifers are infected with black rot disease e.g., cauliflower, cabbage, Brussels, sprout etc. (Vicente et al., 2001). The nucleotide BLAST analysis of 16S rRNA, AvrBs1, AvrGf1 showed a 100% identity with different Xcc strains reported from Germany (B100; AM920689), Brazil (ATCC 33913; AE008922), India (Xcc-C7; CP077958), France (CFBP 5817; CM002673) and China (8004; CP000050) (Singh et al. 2022). Whilst, the nBLAST analysis of xcc-b100_4389 showed 100% nucleotide identity with Xcc race 1 (B100; AM920689), Germany. The sequences were deposited in GenBank (16S rRNA: OM839780; AvrBs1: OM994397; AvrGf1: OM994398; xcc-b100_4389: OM994399). The XccAK1 strain (ITCCBH_0014) was deposited in Indian Type Culture Collection, ICAR-IARI, New Delhi, India. Presently, it is a first report of necrotic black rot on B. juncea cv. Pusa Bold incited by Xcc race 1, India. Previous research reported the black rot disease on other species of the Brassica genus e.g., B. oleracea, and B. napus in Serbia (Popovic et al., 2013) and Argentina (Gaetan et al., 2005).

Mucilaginibacter Phenanthrenivorans sp. nov., a Novel Phenanthrene Degradation Bacterium Isolated from Wetland Soil

BJC16-A38T, a Gram-negative, aerobic and non-motile rod-shaped strain was isolated from a permafrost wetland soil sample. BJC16-A38T was oxidase- and catalase-positive, and produced pale yellow colonies on modified R2A agar plates. The 16S rRNA gene sequence of BJC16-A38T shared the highest sequence similarity with those of Mucilaginibacter xinganensis BJC16-A31T (97.44%), Mucilaginibacter gotjawali SA3-7T (96.79%) and Mucilaginibacter frigoritolerans FT22T (96.14%). Phylogenetic analysis revealed that BJC16-A38T formed a separate lineage together with strain M. xinganensis BJC16-A31T in the genus Mucilaginibacter. BJC16-A38T contained menaquinone-7 (MK-7) as the predominant isoprenoid quinine. Major fatty acids in cells were iso-C15:0, summed feature 3 (16:1ω7c/16:1ω6c) and iso-C17:03-OH. BJC16-A38T contained phosphatidylethanolamine, two unknown polar lipids, six unidentified phospholipids and an unidentified aminolipid. The Genome of BJC16-A38T was sequenced using the Genome Analyzer IIx sequence platform and 38 contigs were produced in total with an average G + C percentage of 44.00%. The average nucleotide identity (ANI) of BJC16-A38T with respect to those of M. xinganensis BJC16-A31T, M. gotjawali SA3-7T and M. frigoritolerans FT22T were 79.60%, 77.24% and 77.58%, respectively. Digital DNA-DNA hybridization (DDH) values between BJC16-A38T and the tree reference strains were 21.30%, 19.60% and 19.70%, respectively. BJC16-A38T exhibited phenanthrene biodegradation activity that can degrade 88.02% phenanthrene in the MM medium after 7days cultivation. Phenotypic, chemotaxonomic, phylogenetic and genomic characteristics concluded that strain BJC16-A38T represents a novel species of the genus Mucilaginibacter. Hence, the name Mucilaginibacter phenanthrenivorans sp. nov. is proposed. The type strain is BJC16-A38T (= CGMCC 1.12693T = NBRC 110383T).

P223 First case of Candida auris candidemia in Manipur, Northeast India

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjective Candida auris is known as an emerging ‘superbug’ because of its intrinsic resistance to one or more, sometimes to all available antifungal drugs and spreading globally. It has the ability to cause devastating nosocomial infections. In India, C. auris infection is on the rise with reports from north, south, central and eastern India. Here we present the first case of C. auris fungemia from a tertiary care hospital of Manipur in Northeast India.MethodsA 15-year-old Muslim girl was referred from a private hospital to Regional Institute of Medical Sciences (RIMS) hospital on November 19, 2021 with a history of burning epigastrium, headache, loss of appetite, shortness of breath, dry cough, fever, and generalized weakness for last 3 days. At the time of admission she was cyanotic. Family gave history of congenital heart disease and frequent visits to hospital. Echocardiogram revealed congenital cyanotic heart disease (Tetralogy of Fallot) showing large perimembranous VSD with bidirectional shunt. A complete hemogram showed neutrophilic leukocytosis with shift to left with band form, absolute monocytosis, and increased RBC count with mild anisocytosis. On November 24, 2021, 5 days after admission, her condition deteriorated and she was shifted to ICU. However, the condition of the patient deteriorated and she died on November 29, 2021 due to acute decompensated heart failure. Follow-up of other patients admitted in the same ward revealed no candidemia in next the few weeks.ResultsA single blood culture sent on November 29, 2021 was incubated in an automated blood culture system, BacT Alert and showed growth of budding yeast cells. Growth in SDA revealed it to be Candida sps. and Gram-stained smear examination revealed presence of budding yeast cells but no pseudohyphae. Germ tube test was negative. On CHROM agar, it produces pale yellow colonies at 24 h which progresses to light purple colonies around the rim at 48 h. Further processing in VITEK 2 (Biomerieux) identified it as C. auris. The isolate was sent to National Culture Collection of Pathogenic Fungi, WHO collaborating center, PGIMER and the isolate was confirmed as Candida auris by MALDI-TOF assay.Conclusion Candida auris is spreading irrespective of the level of health care. Blood culture before administration of antibiotics and in febrile sick patients cannot be underestimated. Rapid and accurate identification methods for timely diagnosis and stringent infection control measures with an emphasis on hand hygiene are important to prevent and control C. auris outbreaks.

Solihabitans fulvus gen. nov., sp. nov., a member of the family Pseudonocardiaceae isolated from soil.

A polyphasic taxonomic study was carried out on an actinobacterial strain (AN110305T) isolated from soil sampled in the Republic of Korea. Cells of the strain were Gram-stain-positive, aerobic, non-motile and rod-shaped. Comparative 16S rRNA gene sequence studies showed a clear affiliation of strain AN110305T with Actinomycetia, with highest pairwise sequence similarities to Goodfellowiella coeruleoviolacea DSM 43935T (97.6%), Umezawaea tangerina MK27-91F2T (97.0%), Kutzneria chonburiensis NBRC 110610T (96.9%), Kutzneria buriramensis A-T 1846T (96.8%), Umezawaea endophytica YIM 2047XT (96.8%), Kutzneria albida NRRL B-24060T (96.7%) and Saccharothrix coeruleofusca NRRL B-16115T (96.6%). Cells of strain AN110305T formed pale-yellow colonies on Reasoner's 2A agar. MK-9 (H4) (68%) and MK-10 (H4) (32%) were the predominant menaquinones. Diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethyl ethanolamine, hydroxy-phosphatidylethanolamine, an unidentified aminolipid and an unidentified aminophospholipid were major polar lipids. Iso-C16:0 (24.5%), anteiso-C15:0 (19.3%), anteiso-C17:0 (15.7%) and iso-C15:0 (15.2%) were the major fatty acids and meso-diaminopimelic acid was the pepdidoglycan. The cell-wall sugars were composed of galactose, glucose, mannose and ribose. The genomic DNA G+C content was 70.7 mol%. Based on genotypic and phenotypic data, strain AN110305T could be distinguished from all genera within the family Pseudonocardiaceae and represents a novel genus and species named Solihabitans fulvus gen. nov., sp nov. The type strain is AN110305T (=KCTC 39307T =DSM 103572T).

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.

Comamonas suwonensis sp. nov., isolated from stream water in the Republic of Korea.

A novel bacterial strain, EJ-4T, isolated from stream water collected at Seo-ho in Suwon, Republic of Korea, was characterized based on a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain EJ-4T belonged to the genus Comamonas. The isolate is Gram-stain-negative, non-motile, aerobic, rod-shaped and forms pale yellow colonies on trypticase soy agar. The optimal growth of this strain was observed aerobically at 30 °C, pH 7 and 0.5 % NaCl. The major fatty acids were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c; 39.7 %) and C16 : 0 (32.0 %). The G+C content of strain EJ-4T was 58.4mol %. The average nucleotide identity and digital DNA-DNA hybridization values between strain EJ-4T and Comamoas testosteroni were 91.8 and 31.2 %, respectively. The major polar lipids detected in the isolate were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The predominant isoprenoid quinone was ubiquinone-8. Based on the results of polyphasic taxonomic analysis of strain EJ-4T, we describe a novel species of the genus Comamonas, for which the name Comamonas suwonensis sp. nov. has been proposed, with EJ-4T (=KCTC 82074T=JCM 34179T=KEMB 1602-279T) as the type strain.

Оccurrence of bacterial spot caused by Xanthomonas arboricola pv. pruni on peach and apricot in Serbia.

Xanthomonas arboricola pv. pruni (Xap) causes bacterial spot of leaves and fruits, defoliation, fruit dropping and twigs, branches or trunk cankers in most cultivated and ornamental Prunus species. The bacterium is listed as an EPPO (European and Mediterranean Plant Protection Organisation) A2 quarantine pathogen. Xap was first detected in 2019 on peach [Prunus persica L., unknown cultivar (cv.)] leaves in a 13-year-old orchard located in Irig (GPS: 45°6'10.538'' N, 19°54'8.04'' E), with a disease incidence of 10-20%. Thereafter, Xap was detected in 2020 on apricot (Prunus armeniaca L., cvs. NS4, NS Rodna and Roxana) leaves and fruits in a 5-year-old orchard located in Bešenovo (GPS: 45°04'59.0'' N, 19°41'23.0'' E), with disease incidence of 30-50%. Symptoms on leaves appeared along leaf midribs or margins in form of brown to black spots, with a pale green to yellow halo, evident on both leaf surfaces. The diseased area on leaves dropped out giving a shot-hole appearance, leaves turn yellow and drop prematurely. Bacterial spots of apricot fruits appeared in form of water-soaked or dark brown sunken lesions. Primarly detection of Xap in collected samples was obtained using polymerase chain reaction (PCR) with Xap - specific primers XapY17-F/XapY17-R, after amplifying target fragments of 943-bp (1). Xap reference strain NCPPB 3156 served as positive control. Isolations performed on yeast extract-dextrose-calcium carbonate agar (YDC) resulted in pale yellow, circular, raised and mucoid colonies after 3 days at 26 °C. A total of 20 representative isolates were aerobic, gram negative, catalase positive, oxidase and arginine dihydrolase negative; they induced hypersensitivity reaction, reduced nitrates, hydrolyzed aesculin and gelatine, but not starch, produced H2S, not produced indole (2). NCBI BLASTn search of the ftsX (ABC transporter ATP-binding protein) sequences (1) indicated 100% identity of Serbian isolates with Xap strains XAP HU2 (P. persica, Acc. no. MG049921) and XAP HU1 (P. armeniaca, Acc. no. KY039173) from Hungary. The nucleotide sequences of one isolate from peach (Xp219) and one from apricot (Xp320) were deposited in GenBank under Acc. nos. MT890969 and MT890970, respectively. Pathogenicity was performed on detached peach and apricot leaves for all 20 isolates and on leaves and shoots of potted 1-year-old plants of peach (cv. Vineyard peach) and apricot (cv. NS Rodna) for three isolates from each host (3, 4). Sterile distilled water and reference strain NCPPB 3156 were used as negative and positive controls. On detached leaves all isolates caused typical water-soaked spots 3 days after inoculation (DAI), while 10 DAI spots became brown and necrotic. Same symptoms were appeared on the leaves of potted plants. On peach and apricot shoots water-soaked, slightly reddish lesions emerged on inoculation sites 7-10 DAI, while 20 DAI lesions become dark, circular to elliptical 3.5-4 and 2.5-3 cm in size for peach and apricot, respectively. Negative controls were symptomless. Reisolated bacteria were confirmed to be the same as the original using PCR (1), fulfilling Koch's postulates. This is the first report of Xap in Serbia, which has occurred with a limited distribution in the Fruška Gora region (Vojvodina). Only two orchards in Serbia have been deteched with Xap so far. In the diseased peach orchard Xap was eradicated by uprooting trees. The apricot orchard is still under official control to limit disease spread. Appropriate cultivation practices, national inspection and surveillance is in place to prevent further pathogen spread and establishment to new hosts and regions in Serbia.

Pseudomonas brassicae sp. nov., a pathogen causing head rot of broccoli in Japan.

Phytopathogenic bacteria, MAFF 212426, MAFF 212427T, MAFF 212428 and MAFF 212429, were isolated from head rot lesions of broccoli (Brassica oleracea L. var. italica Plenck) in Hokkaido, Japan, and subjected to polyphasic taxonomic characterization. The cells were Gram-reaction-negative, aerobic, non-spore-forming, motile with one or two polar flagella, rod-shaped and formed pale yellow colonies. Results of 16S rRNA gene sequence analysis showed that they belong to the genus Pseudomonas with the highest similarity to 'Pseudomonas qingdaonensis' JJ3T (99.86 %), Pseudomonas laurentiana GSL-010T (99.22 %), Pseudomonas huaxiensis WCHPs060044T (99.01 %), Pseudomonas japonica NBRC 103040T (98.87 %) and Pseudomonas alkylphenolica KL28T (98.73 %). The genomic DNA G+C content was 63.4 mol% and the major fatty acids (>5 % of the total fatty acids) were summed feature 3 (C16 : 1 ω7c / C16 : 1 ω6c), C16 : 0, summed feature 8 (C18 : 1 ω7c / C18 : 1 ω6c) and C17 : 0 cyclo. Multilocus sequence analysis using the partial rpoD, gyrB and rpoB gene sequences and phylogenomic analyses based on the whole genome sequences demonstrated that the strains are members of the Pseudomonas putida group, but form a monophyletic, robust clade separated from their closest relatives. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values corroborated their novel species status, with 88.39 % (ANI) and 35.8 % (dDDH) as the highest scores with 'P. qingdaonensis' JJ3T. The strains were differentiated from their closest relatives by phenotypic characteristics, pathogenicity on broccoli, and whole-cell MALDI-TOF mass spectrometry profiles. The phenotypic, chemotaxonomic and genotypic data showed that the strains represent a novel Pseudomonas species, for which the name Pseudomonas brassicae sp. nov. is proposed. The type strain is MAFF 212427T (=ICMP 23635T).

Pseudomonas salomonii, another causal agent of garlic spring rot in Japan

In the 2010s, a sporadic garlic disease in Hokkaido and Kagawa, Japan caused browning and withering of leaves, collapse of aboveground parts, and discoloration of papery sheathing leaves of bulbs and cloves, consistent with the description of spring rot disease. However, the typical rot of storage leaves of cloves was not found. From diseased tissues, bacteria that formed pale-yellow colonies were isolated and demonstrated to be pathogenic on garlic after inoculation and reisolation of the isolates. The gram-negative, aerobic rods with one or two polar flagella were identified as Pseudomonas salomonii based on biochemical and physiological characterizations, PCR assays, phylogenetic analyses, and MALDI-TOF MS analysis. To the best of our knowledge, this study is the first report of P. salomonii in Japan. P. salomonii was originally described in France as the causal agent of cafe au lait disease of garlic, which can be considered as “a type of spring rot disease that does not rot storage leaves.” Therefore, we are treating the disease caused by P. salomonii in Hokkaido and Kagawa as “spring rot disease”.

Occurrence of Xanthomonas arboricola pv. pruni Causing Bacterial Leaf Spot and Shot-Hole on Peach in Montenegro

During May and June 2019, bacterial leaf spot was observed on peach (Prunus persica L. Batsch) in a young plantation (2017, 2018) on four different cultivars (Sugar Time, Britney Lane, Royal Bell, and Royal Time) in the Podgorica region of Montenegro. Diseased trees appeared sporadically, and incidence of symptomatic leaves per infected tree was 10 to 15%. Angular water-soaked spots surrounded by chlorotic tissue were observed along leaf midribs or margins, turning light brown in later stages. Necrotic lesions sometimes dropped out, giving the leaves a “shot-hole” appearance. When spots coalesced, large areas of necrotic foliar tissue formed. Eventually leaves turned yellow, resulting in premature defoliation. Margins taken from healthy tissue and necrotic lesions were homogenized in sterile distilled water (SDW) and spread onto YDC agar (Schaad et al. 2001), on which mostly pale yellow, translucent, circular, raised, and mucoid Xanthomonas-like colonies were observed after 3 days incubation at 26°C. Twelve representative isolates coded as Xp1 to 12 (Sugar Time, Xp1 to 3; Britney Lane, Xp4 to 6; Royal Bell, Xp7 to 9; and Royal Time, Xp10 to 12) were aerobic, gram negative, catalase positive, and oxidase and arginine dihydrolase negative; hydrolyzed esculin and gelatin but not starch; produced H₂S but not indole; and did not reduce nitrate (Schaad et al. 2001). All reactions corresponded with the reactions of the reference strain NCPPB 3156 of Xanthomonas arboricola pv. pruni (Xap), causal agent of leaf spot, shot-hole, and canker of stone fruit. The identity of the isolates was confirmed by PCR using the Xap-specific primer pair XapY17-F/XapY17-R for the ABC transporter ATP-binding protein (Pothier et al. 2011). A single unique target band of 943 bp was amplified for all isolates tested. Furthermore, sequencing of the housekeeping genes fyuA, rpoD, and gyrB (Young et al. 2008) showed 100% identity of tested isolates with genomic sequences of Xap strains deposited in the NCBI database (CFBP 411, 3921, 5580, 6653, 7099, and 7100) based on BLAST analysis and a neighbor-joining phylogenetic tree. The sequences of four isolates representing each cultivar were deposited in GenBank under accession numbers MN520623 to 26 for fyuA, MN520627 to 30 for rpoD, and MN520631 to 34 for gyrB gene for Xp1, Xp4, Xp7, and Xp10, respectively. Pathogenicity was confirmed on (i) young, fully expanded, healthy, detached peach leaves using a hypodermic syringe without a needle, pressed against the abaxial side of the leaf (bacterial suspension at 10⁷ CFU/ml from a 72-h YDC culture) and (ii) immature fruits stabbed and pressing the bacterial suspension (10⁷ CFU/ml) until overflow. Xap reference strain NCPPB 3156 served as a positive and SDW as a negative control. Inoculated leaves were kept under controlled conditions at 25°C, 16/8-h day/night photoperiod, and 60 to 80% relative humidity. All tested bacterial isolates and the reference strain developed water-soaked spots on leaves 3 days and on fruits 4 days after inoculation. Ten days after inoculation the spots became dark brown and necrotic. SDW-treated controls were negative. Xap was successfully reisolated and confirmed to be Xap using PCR (XapY17-F/XapY17-R). This study demonstrates Xap on peach as a new host in Montenegro. Xap was previously reported from other (15 of 44) European countries (https://gd.eppo.int/taxon/XANTPR/distribution), mainly on peach and plum, and from Montenegro on almond (Panic et al. 1998). As the causal agent of a major disease of stone fruits, Xap is a quarantine organism in Europe (EPPO A2 List of Pests recommended for regulation for EU). Therefore, it is necessary to implement containment and/or eradication measures to prevent further spread to new areas and/or new hosts. Furthermore, a nation-wide survey on the (re)occurrence of Xap in other hosts such as almond (reported by Panic et al. 1998), apricot, and plum advised.

First Report of Necrotic Disease Caused by Pantoea agglomerans on Plum (Prunus salicina) in China

Prunus salicina L., commonly called Chinese plum or Japanese plum, is a small deciduous tree native to China and widely distributed in the world. Since spring of 2018, an unknown disease has affected commercial plums grown in an orchard located in Dazhou, Sichuan Province, China (31°40′41″N, 107°43′57″E). Disease symptoms in plum trees include water-soaked lesions on immature fruits, brown necrotic spots, and holes with a yellow halo in leaves, as well as dark brown oily cankers on twigs. To isolate the causal agent, surface-disinfested tissues from three separate symptomatic fruits, leaves, and twigs were placed onto nutrient agar (NA) plates and were incubated at 28°C. Pale yellow colonies with smooth margins and mucoid texture appeared after 12 h of incubation. Individual colonies were transferred two times to NA plates using the conventional streak plate techniques to obtain pure cultures. Purified bacteria cells were rod shaped, 1.5 to 3.0 μm long, and 0.5 to 1.0 μm wide. Three isolates (named CdjP1, CdjP2, and CdjP3) were used for further characterization. Biochemical tests using Gram stain, MacConkey agar, and Kovacs’s indole kit (Solarbio, Beijing, China) indicated that the three isolates were gram negative, lactose positive, and indole negative. The 16s rDNA fragment was amplified for the three strains using universal primers 27F and 1492R (Lane 1991) and sequenced (GenBank accession nos. MN542182, MN542183, and MN542184). BLASTn analysis revealed the nearly full-length (about 1,400 bp) 16S rDNA sequences of CdjP1, CdjP2, and CdjP3 shared over 99% identity with Pantoea agglomerans (MH101508) (identities 1,400/1,406, 1,407/1,412, 1,405/1,413), P. vagans (CP038853) (identities 1,397/1,406, 1,403/1,412, 1,402/1,414), and P. ananatis (KC178592) (identities 1,399/1,406, 1,405/1,412, 1,404/1,414), respectively. Multilocus sequence analysis has been shown to be a powerful molecular method for delineation of Pantoea species (Deletoile et al. 2009; Tambong et al. 2014). To identify the species of CdjP1, CdjP2, and CdjP3, six housekeeping genes (fusA, gyrB, leuS, pyrG, rlpB, and rpoB) of each strain were amplified and sequenced (GenBank accession nos. MN549363 to MN549380). Then a phylogenetic tree was constructed using Bayes inference methods based on fusA-gyrB-leuS-pyrG-rlpB-rpoB concatenated fragments (2,997 bp), which showed that CdjP1, CdjP2, and CdjP3 are most closely related to P. agglomerans. To fulfill Koch’s postulates, pathogenicity tests were conducted on 3-year-old plum trees (cv. Qinba) grown in Sichuan Province, China (31°40′53″N, 107°44′2″E). Immature plum fruits, leaves, and twigs were wounded with a sterile toothpick and were inoculated with 20 µl of bacterial suspension (10⁸ CFU/ml) of each isolate. Plants inoculated with sterile water were used as a control. All treatments and the control were repeated three times. The trees were then grown under natural conditions (monthly mean maximum temperature 32°C, monthly mean minimum temperature 23°C). After 2 weeks, all test-isolate-inoculated plum fruits, leaves, and twigs developed symptoms similar to those of the natural infections observed. No lesions were observed on the controls. Bacteria reisolated from the infected plum fruits, leaves, and twigs were similar in colony appearance to the original isolates, and the 16s rDNA fragments were identical to their original isolate sequences, thereby fulfilling Koch’s postulates. Based on colony morphology, biochemical tests, 16S rRNA gene sequencing data, and phylogenetic analysis, the pathogen was identified as P. agglomerans. In China, P. agglomerans has been reported to infect cotton, walnut, Vigna angularis, and Ziziphus jujuba (Lu et al. 2015; Ren et al. 2008; She et al. 2019; Yang et al. 2011). To our knowledge, this is the first report of P. agglomerans causing bacterial necrosis of plum in China or worldwide. This report expands the host range of P. agglomerans and will help to develop effective disease control strategies, as the etiology is known.

First Report of Fusarium oxysporum and Fusarium redolens Causing Wilting and Yellowing of Wild Rocket (Diplotaxis tenuifolia) in the United Kingdom

In summer 2015, a UK grower reported crop losses of 20% (>£170k) in glasshouse-grown wild rocket (Diplotaxis tenuifolia, cvs. Voyager and Napoli) with disease symptoms initially seen as chlorosis of leaf veins followed by wilting, yellowing and plant death. Diseased plants were collected from three areas of a glasshouse with further samples taken from two areas of another glasshouse in 2017. Roots were washed, surface sterilized in 70% ethanol for 10s, rinsed twice in sterile water and 5mm sections placed on potato dextrose agar (PDA) containing 20 µg/ml chlorotetracycline. Following incubation at 20°C for 4 days, fungal colonies were sub-cultured onto PDA and examined after 14 days. All colonies resembled Fusarium with four morphotypes identified. Morphotype 1: flat, white colonies, abundant oval to cylindrical microconidia (9.4 x 4.6 µm), no macroconidia. Morphotype 2: pale yellow colonies which darkened with age to orange/brown, no microconidia, abundant macroconidia (33.3 x 5.7 µm), 3-7 septate, dorsiventral curvature, hooked basal cell and papillate apical cell. Morphotype 3: white colonies, cotton-like aerial mycelium with pink pigmentation on the underside. Abundant cylindrical (occasionally oval) microconidia (8.9 x 3.6 µm), sparse macroconidia (15.2 x 4.1 µm), slightly curved and slender, 3 septate with hooked basal cell and pointed apical cell. Morphotype 4: white colonies, cotton like aerial mycelium with orange/red pigmentation on the underside, abundant cylindrical microconidia (8.2 x 3.3 µm), macroconidia extremely sparse (15.4 x 3.9 µm), slightly curved and slender, 3-4 septate with hooked basal cell and pointed apical cell. Morphotype 1 was most frequently isolated from diseased plants. DNA was extracted from cultures of each morphotype (19 x type 1, 4 x type 2, 1 x type 3 and 4) and PCR carried out to amplify and sequence part of the translation elongation factor 1-α (EF1-α) gene (Taylor et al., 2016). Following BLAST searches and phylogenetic reconstruction (Fig. S2), isolates were identified as Fusarium redolens (morphotype 1, GenBank accession no. MK172061), Fusarium equiseti (morphotype 2, MK172060) and Fusarium oxysporum (morphotype 3, MK172059 and type 4, MK172058). F. oxysporum isolates were only identified in 2015. The pathogenicity of each Fusarium sp. was determined on six wild rocket cultivars. Roots of five week-old plants (eight plants per isolate/cultivar combination) were trimmed by 2-3mm and soaked in 100 ml of conidial suspension (1 x 106 cfu/ml) for 10 minutes before transplanting into compost in 9 cm pots. Control plants were dipped in sterile water. Pots were placed in a glasshouse (27°C day, 20°C night) and disease symptoms recorded twice weekly. Typical symptoms of wilting and yellowing (particularly in leaf veins) were observed 12 days after inoculation with F. oxysporum and F. redolens, while uninoculated control plants and those inoculated with F. equiseti remained healthy. Fusarium was consistently re-isolated from symptomatic plants, and following DNA extraction, PCR and sequencing of the EF1-α gene, sequences matched the identity of the inoculated isolate, thus fulfilling Koch’s postulates. This is the first report of Fusarium wilt of rocket in the UK and to our knowledge the first ever report of F. redolens causing wilt of wild rocket. F. oxysporum has been reported to cause wilt of wild rocket in Italy (Garibaldi et al. 2003) whilst F. redolens is known to cause wilt of chickpea (Jimenez-Fernandez et al. 2011).

Jatrophihabitans telluris sp. nov., isolated from sediment soil of lava forest wetlands and the emended description of the genus Jatrophihabitans.

A novel actinobacterial strain, designated N237T, was isolated from sediment soil of wetlands at Meonmulkkak, Dongbaek-Dongsan, the lava forest, Gotjawal, Jeju, Republic of Korea. Cells of strain N237T were Gram-stain-positive, non-motile rods and formed pale yellow colonies on ten-fold diluted Reasoner's 2A agar. Strain N237T contained iso-C16 : 0 and C17 : 1ω8c as the major fatty acids, MK-9(H4) as the predominant isoprenoid quinone and meso-DAP as the diamino acid in the peptidoglycan. It contained diphosphatidylglycerol, phosphatidylinositol polymannosides, an unidentified phospholipid, an unidentified aminophospholipid, an unidentified aminolipid, two unidentified glycophospholipids, three unidentified glycolipids and two unidentified lipids as polar lipids. The DNA G+C content was 68.1 mol%. Strain N237T formed a separate lineage in the genus Jatrophihabitans, as demonstrated by phylogenetic analysis based on 16S rRNA sequencing. It was most closely related to Jatrophihabitans soli KIS75-12T (95.6 % sequence similarity). The combined phenotypic, chemotaxonomic and phylogenetic characteristics supported the conclusion that strain N237T represents a novel species in the genus Jatrophihabitans, for which the name Jatrophihabitans telluris sp. nov. is proposed. The type strain is N237T (=KCTC 39922T=NRRL B-65477T).

First Report in China on the Identification and Drug Sensitivity of Mycobacterium elephantis Isolated from the Milk of a Cow with Mastitis

ObjectiveIn this study, milk from a cow with mastitis was analyzed to determine the presence of mycobacterial infection. Milk quality and security problems pertaining to the safe consumption of dairy products were also discussed in this study. MethodsMilk was preprocessed with 4% NaOH. Then, mycobacteria were isolated from the milk sample on L-J medium. The isolate was identified using multiple loci Polymerase Chain Reaction (PCR) and multi-locus sequence analysis with 16S rRNA, sodA, hsp65, and ITS genes. The drug sensitivity of the isolate to 27 antibiotics was tested through alamar blue assay. ResultsSmooth, moist, pale yellow colonies appeared on the L-J medium within a week after inoculation. Based on the results of multiple loci PCR analysis, the isolate was preliminarily identified as non-tuberculous mycobacteria. The 16S rRNA, SodA, hsp65, and ITS gene sequences of the isolate exhibited 99%, 99%, 99%, and 100% similarities, respectively, with those of the published reference strains of Mycobacterium elephantis (M. elephantis). The drug sensitivity results showed that the strain is resistant to isoniazid, p-aminosalicylic acid, and trimesulf but is sensitive to ofloxacin, rifampicin, amikacin, capreomycin, moxifloxacin, kanamycin, levofloxacin, cycloserine, ethambutol, streptomycin, tobramycin, rifabutin, ciprofloxacin, linezolid, cefoxitin, clarithromycin, and minocycline. ConclusionTo the best of our knowledge, this study is initially to report the isolation of M. elephantis from the milk of a cow with mastitis in China.