Xcc Isolates Research Articles

Genetic fingerprinting distinguishes pathogenic variability of asiatic citrus canker pathogen, Xanthomonas citri pv. citri type A

Citrus canker, caused by Xanthomonas citri pv. citri (Xcc), is one of the most widespread citrus disease. In this study, 54 isolates of Xcc were obtained from citrus canker-infected leaves, twigs and fruit samples collected from various citrus-growing regions in Bangladesh. These isolates were analysed through pathogenicity tests and PCR amplification. The results revealed varying levels of aggressiveness among the isolates, ranging from weak to moderate and aggressive. No correlation was found between the host plants and their geographical locations. The genetic analysis of Xanthomonas isolates from around the world revealed 100 % similarity. All the Xanthomonas isolates were grouped into 2 major clusters. DNA fingerprinting showed high genetic variability, with no correlation between hosts, geographic locations or aggressiveness levels. This information on the population diversity of the citrus canker pathogen, Xanthomonas citri pv. citri (Xcc), will be valuable for developing sustainable management strategies for citrus cankers in Bangladesh and the broader Indian sub-continent region.

Complete genome sequence of Xanthomonas phage M29, a new member of Foxunavirus isolated in the Czech Republic.

The newly discovered Xanthomonas phage M29 (Xp M29) is the first lytic phage infecting Xanthomonas campestris pv. campestris (Xcc) that was isolated from cabbage leaves in the Czech Republic. The phage consists of icosahedral head approximately 60nm in diameter and a probably contractile tail of 170nm. The complete genome size was 42891bp, with a G + C content of 59.6%, and 69 ORFs were predicted on both strands. Pairwise nucleotide comparison showed the highest similarity with the recently described Xanthomonas phage FoX3 (91.2%). Bacteriophage Xp M29 has a narrow host range infecting 5 out of 21 isolates of Xcc. Xp M29 is a novel species in a newly formed genus Foxunavirus assigned directly to the class Caudoviricetes.

A promoter trap in transgenic citrus mediates recognition of a broad spectrum of Xanthomonas citri pv. citri TALEs, including in planta-evolved derivatives.

Citrus bacterial canker (CBC), caused by Xanthomonas citri subsp. citri (Xcc), causes dramatic losses to the citrus industry worldwide. Transcription activator-like effectors (TALEs), which bind to effector binding elements (EBEs) in host promoters and activate transcription of downstream host genes, contribute significantly to Xcc virulence. The discovery of the biochemical context for the binding of TALEs to matching EBE motifs, an interaction commonly referred to as the TALE code, enabled the in silico prediction of EBEs for each TALE protein. Using the TALE code, we engineered a synthetic resistance (R) gene, called the Xcc-TALE-trap, in which 14 tandemly arranged EBEs, each capable of autonomously recognizing a particular Xcc TALE, drive the expression of Xanthomonas avrGf2, which encodes a bacterial effector that induces plant cell death. Analysis of a corresponding transgenic Duncan grapefruit showed that transcription of the cell death-inducing executor gene, avrGf2, was strictly TALE-dependent and could be activated by several different Xcc TALE proteins. Evaluation of Xcc strains from different continents showed that the Xcc-TALE-trap mediates resistance to this global panel of Xcc isolates. We also studied in planta-evolved TALEs (eTALEs) with novel DNA-binding domains and found that these eTALEs also activate the Xcc-TALE-trap, suggesting that the Xcc-TALE-trap is likely to confer durable resistance to Xcc. Finally, we show that the Xcc-TALE-trap confers resistance not only in laboratory infection assays but also in more agriculturally relevant field studies. In conclusion, transgenic plants containing the Xcc-TALE-trap offer a promising sustainable approach to control CBC.

Genome Sequences of Four Isolates of Xanthomonas campestris pv. campestris, the Causal Agent of Black Rot

Black rot caused by Xanthomonas campestris pv. campestris ( Xcc) poses a threat to cruciferous crops worldwide. Here, we report the genome sequences of four Xcc isolates, which were assembled using a combination of Nanopore long-read and MGISEQ-2000 short-read sequences. The assembled genomes of the isolates collected in Dingxi (Gansu Province), Ji'nan (Shandong Province), and Yixing (Jiangsu Province) are 5,127,125, 5,124,023, and 5,088,826 bp in size, respectively, and harbor 4,683, 4,680, and 4,608 protein-coding genes, respectively, whereas the isolate collected in Tianjin (Tianjin Municipality) has a chromosome of 5,131,420 bp and a plasmid of 38,762 bp, harboring 4,684 and 48 protein-coding genes, respectively. These genome data provide valuable resources for studies that will enrich our understanding of genomic evolution and genetic diversity of Xcc isolates and the molecular basis of Xcc-host interactions, thereby assisting the development of novel strategies for effectively controlling the disease caused by Xcc. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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).

STUDY OF XANTHOMONAS CAMPESTRIS PV. CAMPESTRIS ISOLATES ORIGINATING FROM SOIL AND BRASSICA SPP. SEEDS

Xanthomonas campestris pv. campestris (Xcc) is a harmful pathogen of the Brassicaceae agricultural plants in Serbian agroecological conditions. The bacterium survives in soil, plant residues, seeds, and wild and weedy plants. This study aimed to characterize Xcc isolates that persist in field soil and roots where they represent a risk to causing disease of Brassicaceae plants. Collection of soil samples was carried out in the period 2015 - 2017 in four locations regions Vojvodina with cabbage cultivation in monoculture or crop rotations. The cabbage seed samples were collected in the same period in Futog and Despotovo locations (agricultural farms) and local farmer markets. All isolations were done on SX and YDC agar plates with the addition of cycloheximide at 250 mg/l. Based on the pathogenicity tests and morphological and biochemical characteristics, 28 identified Xcc isolates were selected, 12 from soil and 16 from seeds. Molecular confirmation of the Xcc isolates was done using multiplex PCR (m-PCR) with Xcc-specific primer sets DLH 120/125 and Zup 2309/2310. In all selected isolates and the reference Xcc strain NCPPB1144, the expected 370 and 619 bp PCR fragments were amplified. Characterizing isolates by rep-PCR fingerprinting using BOX A1R and (GTG)5 primers resulted in informative patterns. Cluster analysis grouped the strain Xcc NCPPB 1144 with all Xcc isolates, while other tested Xanthomonads were clustered separately. Based on the similarity level of 90%, we obtained three out of 12 representative isolates from soil and four out of 16 representative seed isolates. The characterization of Xcc isolates from soil and seeds will enable their comparison with those pathogenic isolated from leaves of Brassicaceae plants.

Comparative Genomic Analysis of Xanthomonas campestris pv. campestris Isolates BJSJQ20200612 and GSXT20191014 Provides Novel Insights Into Their Genetic Variability and Virulence.

Black rot is a disease that has a severe impact on cabbage yield and quality in China. Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot of Brassicaceae crops. So far, the whole genomic sequences of more than 30 Xcc isolates have been sequenced; however, little information about genomic variability and virulence has been reported. In this study, 12 Xcc isolates were isolated from diseased cabbage leaves in seven Chinese provinces and two municipalities from July 2019 to November 2020. Pathogenicity analysis showed that isolate GSXT20191014 was more aggressive than BJSJQ20200612 and HRIW 3811 on cabbage inbred line 1371. Both BJSJQ20200612 and GSXT20191014 were sequenced and comparatively analyzed. The results showed that BJSJQ20200612 and GSXT20191014 have a single circular chromosome comprising 5,115,975 and 4,975,682 bp, respectively. Compared to the other six sequenced strains, 26 and 47 variable genomic regions were found in BJSJQ2020061 and GSXT20191014 genomic sequences, respectively. The variable genomic regions could be responsible for the genetic variation in Xcc strains and have led to the differences in type III secreted effector repertoires, virulence factors and secreted proteins between these two strains. Among the identified secreted proteins, two copies of peptidase S8/S53 were found in GSXT20191014-specific chromosomal segments. The common effectors xopR, xopH, avrBs1, and xopAH are found in most Xcc genomes, but they are absent in the GSXT20191014 genome. Variations in the composition of exopolysaccharides (EPS) and lipopolysaccharides (LPS) may aid GSXT20191014 isolate infections to evade recognition by the host immune system. Our results revealed a direct correlation between genomic variability and Xcc virulence. We also developed several markers for detecting BJSJQ20200612 and GSXT20191014 isolates and further tested the rest of our other 10 isolates. Finally, the isolated Xcc strains were classified into three genetic subgroups by specific molecular markers and multilocus sequence typing (MLST) approach. BJSJQ20200612 and GSXT20191014 isolates were also classified into two subgroups of Xcc according to the core-genome-based phylogenetic tree. This study extended our understanding of Xcc genomic features and provided the foundation to further characterize the mechanisms for Xcc virulence and a clue for black rot resistance breeding.

Characterization of Xanthomonas campestris pv. campestris in Algeria

Xanthomonas campestris pv. campestris (Xcc) causes the black rot of cruciferous plants. This seed-borne bacterium is considered as the most destructive disease to cruciferous crops. Although sources of contamination are various, seeds are the main source of transmission. Typical symptoms of black rot were first observed in 2011 on cabbage and cauliflower fields in the main production areas of Algeria. Leaf samples displaying typical symptoms were collected during 2011 to 2014, and 170 strains were isolated from 45 commercial fields. Xcc isolates were very homogeneous in morphological, physiological and biochemical characteristics similar to reference strains, and gave positive pathogenicity and molecular test results (multiplex PCR with specific primers). This is the first record of Xcc in Algeria. Genetic diversity within the isolates was assessed in comparison with strains isolated elsewhere. A multilocus sequence analysis based on two housekeeping genes (gyrB and rpoD) was carried out on 77 strains representative isolates. The isolates grouped into 20 haplotypes defined with 68 polymorphic sites. The phylogenetic tree obtained showed that Xcc is in two groups, and all Algerian strains clustered in group 1 in three subgroups. No relationships were detected between haplotypes and the origins of the seed lots, the varieties of host cabbage, the years of isolation and agroclimatic regions.

Variation in the aggressiveness of Xanthomonas citri subsp. citri pathotypes A and A* on three Citrus species, and epiphytic growth on some citrus weeds

Citrus bacterial canker, caused by Xanthomonas citri subsp. citri (Xcc), is a widespread disease in citrus-producing areas of the tropics and subtropics. It is a serious threat to most commercial citrus cultivars and species. In this study, aggressiveness of different isolates of Xcc was determined on leaves of Citrus aurantiifolia, Citrus limon ‘Eureka’ and Citrus sinensis, and the population dynamics of isolates of two Xcc pathotypes (A and A*) was evaluated on eight plant species considered weeds of citrus, including Amaranthus caudatus, Medicago sativa, Sorghum halepense, Xanthium strumarium, Portulaca oleracea, Avena ludoviciana, Plantago lanceolata and Chenopodium album under greenhouse conditions. Isolates varied from aggressive to weakly aggressive on the basis of the intensity and persistence of water soaking and the development of necrosis 12 days post-inoculation. Both Xcc pathotypes multiplied on all plant species tested except Portulaca oleracea up to 20 days post-inoculation under greenhouse conditions. A high bacterial growth rate was observed on Medicago sativa and Plantago lanceolata. Strains of the A* pathotype successfully proliferated on all weeds except Portulaca oleracea, attaining a population density even higher than that of the A pathotype. The results clearly suggest that weed management is an important strategy in controlling canker disease in citrus-growing areas.

Efficacy of different copper compounds in the control of Xanthomonas citri subsp. citri pathotypes A and A*

AbstractCitrus canker disease is one of the most devastating diseases that attacks citrus, especially limes in the Southern parts of Iran, and is caused by Xanthomonas citri subsp. citri (Xcc). The efficacy of several formulations of copper compounds including Bordeaux mixture, copper oxychloride and copper sulphate in controlling Xcc in Key lime was estimated in vitro and in planta using artificial inoculation. Specific primers were used to detect copper‐resistant genes copA, copB and copL in 30 isolates of Xcc. The copA and copL genes were present in all isolates, and copB was detected only in 6 strains. In this study, we observed a very good in vitro growth inhibition activity of copper compounds against Xcc pathotype A. S14 strain (pathotype A*) was the sole isolate that grew on media amended with 2/4 mM of Bordeaux mixture, copper oxychloride and copper sulphate. All other strains (pathotype A) failed to grow on media amended with this concentration. Bordeaux mixture exhibited high efficacy in controlling Xcc in both conditions. However, there were no significant differences in the efficacy of copper oxychloride and copper sulphate at 1.2 mM concentration in planta. A significantly minimum canker necrotic spot and highest disease control was achieved with Bordeaux mixture and copper oxychloride. There was a significant difference in disease severity of the type strain LMG9322 (pathotype A) and Xcc strain S14 (pathotype A*). Our experiments showed that Bordeaux mixture exhibited satisfactory efficacy in controlling the causal agent of citrus canker.

Genetic diversity and virulence of Xanthomonas campestris pv. campestris isolates from Brassica napus and six Brassica oleracea crops in Serbia

The present study provides insight into the diversity of 147 Xanthomonas campestris pv. campestris (Xcc) isolates obtained from six Brassica oleracea vegetable crops (broccoli, cabbage, cauliflower, collard greens, kale, kohlrabi) and the winter oilseed rape crop Brassica napus, collected from different regions in Serbia in 2014. The XCF/XCR pathovar‐specific primer set was used for fast preliminary identification. In repetitive sequence‐based PCR (BOX, ERIC and REP) of all isolates, a higher level of genetic diversity was found in winter oilseed rape isolates compared to isolates from the other hosts. ERIC and REP‐PCR showed the highest heterogeneity, with 10 and nine banding patterns, respectively. The REP‐PCR results showed the highest correlation (70%) with those obtained with multilocus sequence analysis (MLSA), performed with 10 housekeeping genes (fusA, gap‐1, gltA, gyrB1, lacF, lepA, rpoD, dnaK, fyuA and gyrB2). Three distinct phylogenetic groups of winter oilseed rape isolates were detected using MLSA. Two genes, gltA and rpoD, showed the greatest ability to identify and discriminate winter oilseed rape Xcc isolates from isolates of the other six hosts. The lepA gene exhibited specific three‐nucleotide changes in sequences of some of the isolates. Results of virulence testing of 18 representative isolates showed statistically significant host–pathogen specialization for Xcc isolates from winter oilseed rape, cauliflower, kale and kohlrabi. In conclusion, oilseed rape isolates are more genetically diverse and show greater specialization to their host in comparison to the rest of the tested isolates from other brassica hosts.

Pan Proteome of Xanthomonas campestris pv. campestris Isolates Contrasting in Virulence.

Fully sequenced genomes of Xanthomonas campestris pv. campestris (Xcc) strains are reported. However, intra-pathovar differences are still intriguing and far from clear. In this work, the contrasting virulence between two isolates of Xcc - Xcc51 (more virulent) and XccY21 (less virulent) is evaluated by determining their pan proteome profiles. The bacteria are grown in NYG and XVM1 (optimal for induction of hrp regulon) broths and collected at the max-exponential growth phase. Shotgun proteomics reveals a total of 329 proteins when Xcc isolates are grown in XVM1. A comparison of both profiles reveals 47 proteins with significant abundance fluctuations, out of which, 39 show an increased abundance in Xcc51 and are mainly involved in virulence/adaptation mechanisms, genetic information processing, and membrane receptor/iron transport systems, such as BfeA, BtuB, Cap, Clp, Dcp, FyuA, GroEs, HpaG, Tig, and OmpP6. Several differential proteins are further analyzed by qRT-PCR, which reveals a similar expression pattern to the protein abundance. The data shed light on the complex Xcc pathogenicity mechanisms and point out a set of proteins related to the higher virulence of Xcc51. This information is essential for the development of more efficient strategies aiming at the control of black rot disease.

Biological and molecular characterization of a bacteriophage infecting Xanthomonas campestris pv. campestris, isolated from brassica fields.

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot of crucifers. Here, we report a virus that infects Xcc isolated from brassica fields in Brazil. Morphological, molecular and phylogenetic analysis indicated that the isolated virus is a new member of the genus Pbunavirus, family Myoviridae, and we propose the name "Xanthomonas virus XC 2" for this virus. The isolated virus has a narrow host range, infecting only Xcc isolates, and it did not infect unrelated bacteria. These results indicate that the isolated bacteriophage is highly specific for Xcc and may be a potential agent for biological control.

Testing of Inoculation Methods and Susceptibility Testing of Perspective Cabbage Breeding Lines (Brassica Oleracea convar. Capitata) to the Black Rot Disease Caused by Xanthomonas Campestris pv. Campestris

The susceptibility of twenty‑four cabbage breeding lines to Xanthomonas campestris pv. campestris was evaluated. The selection of appropriate inoculation method was done on 4 cabbage cultivars (‘Cerox’, ‘Sintex’, ‘Sonja’ and ‘Avak’). One month old plants were infected by 5 inoculation methods (spraying, injection by syringe, multiple pricking, carborundum abrasion and scissor clipping method). Four different bacterial isolates of Xcc (WHRI 3811, 3971A, 1279A; SU) and their mixture were evaluated for the aggressiveness on ‘Cerox’ and ‘Sonja’ cultivars. On the basis of obtained results, breeding lines of head cabbage were inoculated by mixture of all tested isolates using multiple pricking method. The disease severity of inoculated seedlings proved high susceptibility of young plants to the Xcc infection. The disease incidence determined 75 and 105 days after sowing showed changes for 16 of tested lines and indicated that resistance testing should be observed until mature stage. The study revealed five breeding lines (DP25, T1, IT10, Kalibos and Avak1) with disease incidence lower than 20 % as perspective sources of resistance for further breeding.

Whole-Genome Re-Alignment Facilitates Development of Specific Molecular Markers for Races 1 and 4 of Xanthomonas campestris pv. campestris, the Cause of Black Rot Disease in Brassica oleracea.

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc), is a seed borne disease of Brassicaceae. Eleven pathogenic races have been identified based on the phenotype interaction pattern of differential brassica cultivars inoculated with different strains. Race 1 and 4 are the two most frequent races found in Brassica oleracea crops. In this study, a PCR molecular diagnostic tool was developed for the identification of Xcc races 1 and 4 of this pathogen. Whole genomic sequences of races 1, 3, 4 and 9 and sequences of three other Xanthomonas pathovars/species (X. campestris pv. incanae (Xci), X. campestris pv. raphani (Xcr) and X. euvesicatoria (Xev) were aligned to identify variable regions among races. To develop specific markers for races 1 and 4, primers were developed from a region where sequences were dissimilar in other races. Sequence-characterized amplified regions (SCAR) and insertion or deletion of bases (InDel) were used to develop each specific set of primers. The specificity of the selected primers was confirmed by PCR tests using genomic DNA of seven different Xcc races, two strains of X. campestris pathovars and other species of bacteria. Bacterial samples of the races 1 and 4 isolates were collected from artificially inoculated cabbage leaves to conduct bio-PCR. Bio-PCR successfully detected the two Xcc isolates. By using our race-specific markers, a potential race 1 strain from the existing Korean Xcc collection was identified. The Xcc race 1 and 4-specific markers developed in this study are novel and can potentially be used for rapid detection of Xcc races through PCR.

Characterization, genetic diversity and distribution of Xanthomonas campestris pv. campestris races causing black rot disease in cruciferous crops of India

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc) is a disease of crucifer crops. The objective of this study was to characterize races of Xcc, their distribution and genetic diversity in India. Two hundred and seventeen isolates of bacteria were obtained from 12 different black rot‐infected crucifer crops from 19 states of India; these were identified as Xcc based on morphology, hrpF gene and 16S rRNA gene based molecular markers and pathogenicity tests. Characterization of races was performed by using a set of seven differential crucifer hosts, comprising two cultivars of turnip (Brassica rapa var. rapa) and cultivars of Indian mustard (B. juncea), Ethiopian mustard (B. carinata), rapeseed mustard (B. napus), cauliflower (B. oleracea) and Savoy cabbage (B. oleracea var. sabauda). Races 1, 4 and 6 of Xcc were identified and, among these races, race 1 followed by race 4 dominated most of the states of India. Genetic diversity of the Indian isolates of Xcc was analysed using repetitive sequence‐based PCR (rep‐PCR) including primers for REP (repetitive extragenic palindromic), ERIC (enterobacterial repetitive intergenic consensus) and BOX (amplifying with BOX A1 R primer) repetitive elements. This method of fingerprinting grouped the isolates into 56 different DNA types (clusters) with a 75% similarity coefficient. Among these clusters, DNA types 22 and 53 contained two different races 1 and 4, whereas DNA type 12 contained races 1, 4 and 6. However, no clear relationship was observed between fingerprints and races, hosts or geographical origin.

Evaluation of Paenibacillus spp. isolates for the biological control of black rot in Brassica oleracea var. capitata (cabbage)

ABSTRACTThe ability of isolates of Paenibacillus spp. to protect Brassica oleracea var. capitata (cabbage) against the black rot pathogen, Xanthomonas campestris pv. campestris (Xcc),was evaluated. Twenty-four isolates of Paenibacillus spp., isolated from New Zealand-grown brassica hosts or soil, were evaluated for in vitro antagonism towards six Xcc isolates. Seven Paenibacillus spp. isolates with different levels of in vitro suppressive activity against Xcc were screened in pot experiments for their capacity to reduce black rot symptoms on cabbage. Two Paenibacillus isolates (P10 and P16) exhibited biocontrol activity against Xcc, and four isolates (P1, P6, P9, and P24) reduced cabbage seed germination and seedling emergence. The dependence of bioactivity on inoculum rate was investigated with three Paenibacillus isolates (P6, P10, and P16) at three different concentrations (5 × 108, 5 × 109, and 5 × 1010 CFU ml−1). Negative effects on seedling emergence were detected with isolate P6 at concentrations ≥5 × 109 CFU ml−1. All three isolates applied at the three concentrations reduced black rot symptoms on the cotyledons and true leaves. There was poor or no relationship between the inhibitory effect of Paenibacillus spp. isolates on the growth of Xcc in vitro, and their biocontrol activity in vivo. Paenibacillus isolate P16 was identified as a potential biological control of black rot in cabbage.

Bacterial Secretome Analysis in Hunt for Novel Bacteriocins with Ability to Control Xanthomonas citri subsp. Citri.

Xanthomonas citri subsp. citri (Xcc), the causative agent of bacterial citrus canker, has affected citriculture worldwide. Varieties of means have been used to minimize its devastating effects, but no attention has been given to bacteriocins. Here and for the first time, we report the isolation and characterization of two novel bacteriocins. Secretome containing bacteriocins of isolated bacteria was separated via SDS-PAGE. Each isolated protein band was characterized and checked for its efficacy in controlling two pathogenic isolates of Xcc via disk diffusion assay. The effects of varieties of carbon, nitrogen and phosphate sources were evaluated on both bacterial growth and bacteriocin production via Taguchi orthogonal method. The two bacteriocins showed an activity up to 55ºC that were sensitive to proteases suggesting being protein in nature. Analysis of SDS-PAGE purified protein bands of bacterial secretomes with demonstrated potency against Xcc revealed the presence of peptides with relative molecular masses of 16.9 and 17 kDa for Cronobacter and Enterobacter, respectively. Sequence analysis of peptides revealed an HCP1 family VI secretion system homologue for Cronobacter (YP_001439956) and pilin FimA homologue for Enterobacter (CBK85798.1). A Taguchi orthogonal array was also implemented to determine the effect of temperature and eight other chemical factors on bacteriocin production for each bacterium. Two peptides with novel antibacterial activities effective against Xcc were isolated, characterized and conditions were optimized for their higher production.

Genetic diversity of Xanthomonas campestris pv. campestris isolated from Brassica crops using RAPD and Rep-PCR

Xanthomonas campestris pv. campestris (Xcc) is causal agent of black rot disease in crucifers. Genetic diversity, 28 isolates of Xcc collected from cole crops (cauliflower, cabbage, broccoli and knol khol), turnip, brown and yellow mustard crops from northern region of India was performed by using rep-PCR fingerprinting (BOX, REP and ERIC) and random amplified polymorphic DNA (RAPD). Five DNA type groups were formed by using rep-PCR fingerprinting. In RAPD study, six primers showed monomorphic bands and remaining six primers (OPB-10, OPB- 15, UBC-245, D-11, UBC-220 and OPA-11) exhibited polymorphic amplified product in all isolates of Xcc. Xcc- C281 and Xcc-C261 and Xcc-C268 were formed separate group as DNA types, 1, 2, 3, 4, 5 and 6 respectively. Genetic variability was found among the isolates of Xcc based on their hosts. They were categorized into eight groups at DNA typing at ~50% similarity coefficient and formed 8 DNA type groups at ~ 50% similarity coefficient. Eighteen isolates of Xcc were grouped under DNA type 7, whereas in DNA type 8 (4 isolates).

Pathogenic and Genetic Characterization of Xanthomonas campestris Pv. campestris Races Based on Rep-PCR and Multilocus Sequence Analysis

Xanthomonas campestris pv. campestris (Pammel) Dowson (Xcc) is the causal agent of black rot disease of crucifers worldwide. Seventy five isolates of Xcc were collected from 12 agro-climatic regions of India to determine the distribution pattern of races and diversity of the population. Based on pathogenic reaction on seven standard differential crucifers, race 1, 4 & 6 were found to be prevalent. For assessing the pathogenic diversity, forty one cultivars of crucifers comprising seven Brassica and coeno species were inoculated artificially under field conditions. Brassica juncea cultivars (Pusa Bold, Varuna, Pusa Vijay, Pusa Mustard 21 and Pusa Mustard 25) showed resistance against all the strains of Xcc, whereas the Brassica olerecea cultivar Pusa Ageti was found to be resistant to races 1 and 4. Genetic characterization of these 75 strains of Xcc was carried out using rep-PCR (ERIC, REP and BOXPCRs) followed by phylogenetic analysis. The strains of Xcc clustered into 6 groups at 50% similarity coefficient and among these groups, 28 strains of Xcc belonging to races 1, 4 & 6 were clustered together under Group 5. Sequences of the 16S rRNA, hrpF and efP genes of five strains representing the races 1, 4 and 6 were used for multilocus sequence analysis. Based on sequence analysis of 16S rRNA and hrpF genes, the Indian strains were found to be very closely related to the strain Xcc ATCC 33913 (race 3, UK), whereas based on efP sequences, they were found to be closely related to strains race 1 Xcc B100 (Italy) and race 9 Xcc 8004 (UK).