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.

First report of Bacterial Leaf Streak disease of rice caused by Xanthomonas oryzae pv. oryzicola in Ivory Coast

Bacterial Leaf Streak (BLS) of rice caused by Xanthomonas oryzae pv. oryzicola (Xoc) is considered as the third emerging infectious disease of rice in Africa. First reported in Africa in the 1980s, the disease is now present in at least eight African countries including Burundi, Burkina Faso, Kenya, Madagascar, Mali, Nigeria, Senegal and Uganda. Yield loss caused by BLS is estimated at 20 to 30% (Sileshi and Gebeyehu, 2021). To our knowledge BLS has so far not been reported in Ivory Coast. While BLS has not been described in the adjacent rice-growing countries Ghana and Liberia, Xoc strains isolated from samples collected between 2003 and 2011 in Burkina Faso and Mali have been characterized (Wonni et al., 2014). Xoc is transmitted through rice seeds which favors its spread through rice trading (Sileshi and Gebeyehu, 2021). Given the extensive rice trade between Burkina Faso, Mali and Ivory Coast, we hypothesized that BLS might also be present in this country. Field surveys were carried out across Ivory Coast in October 2018. Typical symptoms of the disease, e.g. translucent lesions in the form of yellow-brown to black streaks with sometimes visible droplets of exudates on the leaf surface, were observed in the area of Korhogo. 5cm-long leaf pieces were successively disinfected, rinsed in sterile water, and then ground using the Qiagen Tissue Lyser System (QIAGEN, Courtaboeuf, France). Leaf powder was resuspended in 1.5 ml of sterile water and incubated at room temperature for 30 minutes. Then, 10 μl of the suspension was streaked on semi-selective PSA medium and incubated at 28 ° C for 3 to 7 days. Colonies characteristic of Xoc, i.e. round, convex, mucous and straw yellow in color were purified from 6 individual samples from 2 distinct sites in Korhogo. To confirm their identity, isolated strains underwent a pathogenicity and molecular characterization test. The multiplex PCR developed for the identification of X. oryzae pathovars (Lang et al., 2010) revealed for all the isolates the characteristic PCR profile of Xoc (two amplicons of 324 and 691 base pairs). Strains of Xoc BLS256 and Xoo PXO99 were used as controls. The pathogenicity test was performed on 5 weeks-old plants of O. sativa cv. Azucena leaves by infiltration with a needleless syringe of a bacterial suspension at an optical density of 0.5. After 7 days of greenhouse incubation (27 ± 1°C with a 12-hour photoperiod), all infiltration points (2 infiltrations x 3 plants per isolate) developed water-soaked lesions identical to the one challenged with BLS256 while water-infiltrated leaves remained asymptomatic. These lesions were collected and subjected to the isolation and multiplex PCR processes described above, thus fulfilling Koch's postulate. Finally, three of the isolates were subjected to sequencing of the housekeeping gene gyrB by PCR amplification using the primers XgyrB1F and XgyrB1R (Young et al., 2008). Analysis of 780bp of the gyrB sequence of strains CI_k1-1, CI_k2-2 and CI_k3-2 revealed 100% identity with the gyrB sequence of Xoc reference strain BLS256 (Acc. No. CP003057) and 10 polymorphic nucleotides compared to the Xoo reference strain PXO99A (Acc. No. CP000967). To our knowledge, this is the first report of BLS in Ivory Coast supported by molecular characterization methods. New surveys in Ivory Coast and neighboring countries where the disease has not been reported will allow to implement collections and assess disease incidence as part of future control strategies.

Phylogeny Analysis of gyrB Gene and 16S rRNA Genes of Pseudomonas aeruginosa Isolated from Iraqi Patients

Infection caused by Pseudomonas aeruginosa is one of the major problems in hospitalized patients which are related to the high mortality. The DNA gyrase B, gyrB reading gene sequence method provides a fast and efficient system for bacterial identification and diagnosis, taxonomic analysis and monitoring of bacteria in the natural environment. Evolution analysis was performed using gene nucleotide sequences for gyrB and 16S rRNA genes. PCR amplifiers were used for the genes under study and their genetic sequences were read. The evolutionary tree was drawn based on the genetic sequences of the classification of P. aeruginosa, compared to the analysis of the 16S rRNA genes, gyrB sequences showed a greater evolutionary deviation of bacteria and may be useful for distinguishing between closely related species. Sequence analysis of 16S rRNA is accurate for identifying unknown bacteria to the genus level. However, the variable gyrB sequence analysis can identify unknown bacteria to the species level. Together with the 16S rRNA analysis, gyrB sequence analysis is considered a useful tool to build the evolutionary relationships of bacteria, especially for the classification of converging bacterial species and controlling the invasive Patho Micobial infection treatment in the hospital.

Genetic heterogeneity amongVibrio alginolyticusstrains, and design of a PCR-based identification method usinggyrBgene sequence

Vibrio alginolyticus, a pathogen among humans and marine animals, is ubiquitous in marine environments. The aims of this study were to analyze the relationships between genetic diversity and origins, and to develop new primers based on the gyrB sequence to identify V. alginolyticus isolated from various sources. To determine the genetic diversity of this bacterium, an arbitrarily primed polymerase chain reaction (AP-PCR) technique was performed on 36 strains of V. alginolyticus isolated from diarrhea patients and from diseased marine animals and environments in southern Thailand. The results showed distinct DNA fingerprints of all strains, indicating that they are genetically heterogeneous. For species-specific identification of V. alginolyticus, primers targeting the gyrB gene of V. alginolyticus were developed. Thirty reference Vibrio spp., 13 non-Vibrio spp., and 160 strains of V. alginolyticus isolated from various sources in southern Thailand were used to evaluate the specificity of these primers. Our results showed that the gyrB primers could specifically identify V. alginolyticus from all sample types. In addition, the detection limit of the PCR was at least 95 pg of DNA template. Therefore, we concluded that the newly designed gyrB primers are rapid, highly sensitive, and specific to identify V. alginolyticus isolated from various sources.

Confirmation of Bacterial Leaf Streak Caused by Xanthomonas oryzae pv. oryzicola on Rice in Madagascar

Bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola is an important disease of rice. BLS is prevalent in Asia and West Africa, where it was first reported in Nigeria and Senegal in the early 1980s (4). Recently, molecular analysis of strains from Mali (2) and Burkina Faso (5) further confirmed the presence of BLS in West Africa. In Madagascar, BLS symptoms were first reported in the 1980s by Buddenhagen but the causal agent was not unequivocally determined (1). To confirm Buddenhagen's observations using modern molecular typing tools, we surveyed several rice fields in the Antananarivo and Antsirabe districts in March 2013. BLS symptoms were observed on cultivated Oryza sativa grown under both upland and lowland conditions, with a proportion of diseased individuals varying from 30% up to 80%. Symptomatic leaves presenting water-soaked lesions that developed into translucent, yellow streaks with visible exudates at the surface were sampled. One to four centimeter long pieces of diseased leaves were ground using the Qiagen TissueLyser system at 30 rps for 30 s (Qiagen, Courtaboeuf, France). The ground tissue was then macerated in 1 ml of sterile water for 1 h at 4°C. Non-diluted and 10-fold diluted tissue macerates were plated on semi-selective PSA medium (peptone 10 g/liter, sucrose 10 g/liter, glutamic acid 1 g/liter, bacto agar 16 g/liter, actidione 50 mg/liter, cephalexin 40 mg/liter, and kasugamycin 20 mg/liter) and incubated for 3 to 7 days at 28°C. Single, yellow, Xanthomonas-like colonies were isolated on non-selective PSA medium. Diagnostic multiplex PCR was performed on single colonies for pathovar identification (3). Five strains that produced three diagnostic bands corresponding to the X. oryzae pv. oryzicola pattern were further analyzed for pathogenicity on 3-week-old O. sativa cv. Nipponbare plants. Bacteria grown on PSA plates and adjusted to 1 × 108 CFU/ml were infiltrated into rice leaves with a needleless 1-ml syringe (2 × 3 infiltrations per plant and strain). Seven days after incubation in the greenhouse (27 ± 1°C with a 12-h photoperiod), inoculated leaves showed water-soaked lesions that produced yellow exudates corresponding to those initially observed in rice fields and observed for leaves challenged with the X. oryzae pv. oryzicola reference strain BLS256. Symptomatic leaf tissues were ground and plated on non-selective PSA medium, resulting in colonies with typical Xanthomonas morphology that were confirmed as X. oryzae pv. oryzicola by multiplex PCR typing (3), thus fulfilling Koch's postulates. Finally, the five strains were subjected to gyrB sequencing upon PCR amplification using the universal primers XgyrB1F (5′-ACGAGTACAACCCGGACAA-3′) and XgyrB1R (5′-CCCATCARGGTGCTGAAGAT-3′). The 743-bp partial gyrB sequences were 100% identical to the gyrB sequence of strain BLS256. As expected, the gyrB sequence of strains KACC10331, MAFF311018, and PXO99A of the X. oryzae pv. oryzae pathovar respectively showed nine, 16, and 10 mismatches in comparison to the Malagasy strains, thus further supporting that they belong to the pathovar oryzicola. References: (1) I. W. Buddenhagen. Int. Rice Comm. Newsl. 34:74, 1985. (2) C. Gonzalez et al. Mol. Plant Microbe Interact. 20:534, 2007. (3) J. M. Lang et al. Plant Dis. 94:311, 2010. (4) D. O. Niño-Liu et al. Mol. Plant Pathol. 7:303, 2006. (5) I. Wonni et al. Plant Dis. 95:72, 2011.

phoRsequences as a phylogenetic marker to differentiate the species in theBacillus subtilisgroup

Bacillus subtilis and its closely related species are indistinguishable from one another by morphological characteristics and 16S rDNA sequences. In this study, the partial phoR sequence was tested to determine the phylogenetic relationship of species in the B. subtilis group. Degenerate primers were developed according to the relatively conserved nucleotide sequences of phoR and the linked gene phoP in the B. subtilis group. The primers amplified a 1100 bp phoR fragment from strains representative of 6 species in the B. subtilis group. Based on the sequenced fragments, 26 type strains comprising these 6 species were clearly distinguished. At the intraspecies level, the phoR sequence similarities were 90%–100%, but at the interspecies level, the phoR sequence similarities were 32.8%–75%. Compared with the gyrB sequence, the phoR sequences showed a larger divergence especially at the interspecies levels. Therefore, the phoR sequence may be an efficient alternative marker for phylogenetic and taxonomic analysis of species in the B. subtilis group. Twenty-three Bacillus undomesticated isolates were tested for identification and phylogenetic analysis based on the phoR and gyrB sequences. The 23 isolates could be clearly delineated into 4 distinct groups, 10 as B. subtilis, 3 as B. mojavensis, 2 as B. atrophaeus, and 8 as B. amyloliquefaciens.