
Citrus blast caused by bacterium
In Mediterranean countries, phytopathogenic bacteria do not generally cause serious damage in citrus groves, excluding the incidence of the blast and black pit, caused by
For the pathogen isolation, 20 samples of necrotic buds from diseased twigs were taken. Small pieces of tissue were taken from the margins of necrotic lesions and were first disinfected with 70% ethanol, then cut and crushed using a mortar and pestle in sterile phosphate-buffered saline (0.27% Na2HPO4, 0.04% NaH2PO4, 0.8% NaCl). After 20 min, the suspensions were plated on King’s medium B (King et al., 1954). After 2 days of incubation at 26°C, a total of 43 fluorescent colonies were selected and purified on Sucrose Nutrient Agar (SNA) (Difco nutrient agar, supplemented with 0.5% sucrose; Difco Laboratories, Detroit, MI, USA) and maintained at −80°C in Luria Bertani medium supplemented with 20% glycerol (Bertani, 1951). The strains CFBP 11 (
Pathogenicity testing of the obtained isolates were checked by artificial inoculation of unripe green lemon (
The lemons were surface sterilized by being dipped into 2% NaClO solution for 2 min and then shortly rinsed with SDW. Four punctures were made on the fruits (1 mm wide, 3 mm deep), by using a sterile hypodermic needle and per 20 μl of the bacterial suspension (106 cfu/ml) was placed on each rupture. Bean pods were inoculated by injection of a bacterial suspension (107 cfu/ml) into young bean pods with a hypodermic syringe. Inoculated fruits and pods were kept in plastic bags and incubated at 25°C for 7 days under conditions of high humidity (70–80%). For each isolate, 12 sites involving three fruits/pods were inoculated. Pathogenic reactions were assessed seven days after inoculation (DAI).
Lilac (
In addition, twigs of mandarin trees were longitudinally ruptured (1 cm) using a sterile scalpel, and per 100 μl of the bacterial suspension (106 cfu/ml) was placed onto the cuts. Twigs were kept in clear polyethylene bags for 24 h under room temperature conditions and high humidity (70%–80%). For each tested isolate and controls, three stems were inoculated. Symptoms were assessed 28 DAI.
Bacterial isolates and reference strains were tested for LOPAT (levan production, oxidase reaction, potato soft rot, arginine dihydrolase activity, and tobacco hypersensitivity) tests (Lelliott and Stead, 1987). Levan production was estimated by growing isolates on SNA medium. For oxidase reaction test, bacteria were grown on SNA medium for 24 h at 25°C until colonies were obtained. Colonies were picked with a loop and rubbed on filter-paper impregnated with 1% (w/v) aqueous tetramethyl-p-phenylene diamine dihydrochloride solution. Production of a purple colour within 10 s was recorded as positive, its development in 10 to 60 s as delayed positive, and the absence of coloration or its still later development as negative (Kovacs, 1956). Pectinase activity was tested by adding bacterial suspension (106 cfu/ml) onto the potato slices and incubated for 24 h at 25°C. Each isolate was tested for arginine dihydrolase production by the method of Thornley (Thornley, 1960), by growing in medium with L-arginine. They were then covered with a 5-mm layer of sterile liquid paraffin, tightly capped, and incubated at 25°C for 7 days. A positive test for the presence of arginine dihydrolase was indicated by a deep pink to red color, whereas a negative test was demonstrated by an absence of color change in the medium. Tobacco hypersensitivity, was tested on leaves of the tobacco. Inoculum were prepared in SDW as described for the pathogenicity test. Reaction to Gram stain, oxiditive/fermentative metabolism of glucose (O/F) and fluorescenent pigment production on the King’s medium B, were also tested (Lelliott and Stead, 1987; Schaad et al., 2001). In addition, GATT tests (Gelatine and Aesculine hydrolysis, formation of Tyrosinase activity and Tartrate metabolism) for the differentiation within the species
Total genomic DNA was extracted using a modification of the procedure of Ausubel et al. (1992). All tested 43 Montenegrian mandarin isolates and
In order to detect the possible presence of the
An enterobacterial repetitive intergenic consensus (ERIC)-PCR with primer pair ERIC1R (5′-ATGTAAGCTCCTGGGGAT TCAC-3′) and ERIC2 (5′-AAGTA AGTGACTGGGGTGAGCG-3′) have been used for differentiation among the isolates (Lupski and Weinstock, 1992). ERIC-PCR conditions were as previously described by de Bruijn (1992). Amplification of PCR was performed with a Mastercycler personal model (Eppendorf) by using the following cycles: 1 initial cycle at 95°C for 7 min; 30 cycles of denaturation at 94°C for 1 min, annealing at 52°C for 1 min, and extension at 65°C for 8 min with a single final extension cycle at 65°C for 15 min and a final soak at 4°C. The PCR amplifications were performed in triplicate. Amplified PCR products were separated by gel electrophoresis on 1% agarose gels in 0.5× TAE buffer for 1 h at 5 V/cm, stained with 0.05 μl/ml ethidium bromide and visualized under UV illumination. Fingerprints generated from different strains were compared visually.
Amplicons for the partial 16S rDNA sequences were generated using universal primers (5′-GAGAGTTTGATCCTGGCTCAG-3′) and P6 (5′-CTACGGCTACCTTGTTACGA-3′) (Grifoni et al., 1995) and reaction conditions described by Scortichini et al. (2005). PCR products were purified with the QIAquick PCR purification kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. The sequencing was performed by Macrogen Inc. (Seoul, Korea) and the sequences were deposited in the GenBank database. The MEGA6 software package (Tamura et al., 2013) was used to align sequences from forward and reverse strands and generate consensus sequences. A basic local alignment search tool BLAST (Altschul et al., 1997) was used to compare unidentified sequences with sequences in public databases. Neighbor joining (NJ) analyses were performed using 16S rDNA sequences which were assembled and edited using FINCHTV v.1.4.0 ( http://www.geospiza.com). Multiple alignments and comparisons with reference strains were performed using CLUSTALW integrated into MEGA6 software (Tamura et al., 2013).
MLSA was performed in order to reveal the relationship between the
In order to estimate the evolutionary relation between
Minimum spanning tree (MST) was generated, where isolates are the nodes of a tree using SplitsTree software (Huson and Bryant, 2006). The method estimates a probability of ancestry for each individual isolate from each of the groups. The individual isolates are assigned to one cluster or jointly to two or more clusters if their genotypes indicate that they were admixed.
During the spring of 2013 and 2014, severe outbreaks of citrus blast were observed in mandarin (cv. Owari) in the regions of Bar and Ulcinj in Montenegro (Fig. 1). Isolations of bacteria from necrotic buds yielded almost pure cultures of whitish, translucent bacterial colonies that were fluorescent on King’s medium B after 48 h of incubation. A total of 43 separate bacterial colonies were selected and purified on SNA for further analysis (Table 1). On SNA, isolates formed large (2–3 mm in diameter), convex (levan positive), cream-whitish, mucoid colonies 2–3 DAI.
All isolates showed similar biochemical and physiological characteristics that were typical of
All isolates were positive for gelatin and aesculin hydrolysis, but negative for tyrosinase activity and metabolism of tartrate, demonstrating typical characteristics for bacteria
PCR amplification with primers B1 and B2 amplified the specific 752 bp band for the
To determine genetic diversity of the 43
Molecular identification of isolates from mandarin was performed using 16SrDNA gene sequencing and MLSA. The sequence analysis of the conserved 16SrDNA gene deposited in NCBI GenBank database under accession numbers KP099969 to KP099980 (Table 2), confirmed that isolates originated from mandarin belong to
The sequences generated with three housekeeping genes (
An MST using the MLSA data using statistical parsimony with split network revealed no ambiguous connections between pathovars and confirmed clustering of testing isolates and reference strains associated within the same genomospecies 1 (Fig. 3). The first cluster (colored in black) included all of our 43 mandarin isolates and members of genomospecies 1. The second cluster included members of genomospecies 2 (colored in blue). The other six clusters present the strains which belong in genomospecies 3, 4 and 6–9 (represented in red, green, light blue, orange, white-green, and white-red, respectively).
The occurrence of citrus blast disease, caused by
The relationship of frost damage and
In our study 43 isolates originated from diseased necrotic mandarin buds were identified and characterized using pathogenicity, biochemical tests and for the first time molecular analysis of pathogen. Based on obtained results, they could be assigned to the phytopathogenic bacterium
Using biochemical and GATT differentiation tests our isolates could be discriminated as
All isolates produced black pit lesions on lemons by needle pricks. The characteristics that positive lesions produced suggest that lemons might be readily inoculated with all strains of this bacteria obtained from any host (Bryan, 1928; Lelliott and Stead, 1987; Smith and Fawcett, 1930). Typical black pit lesions were also formed on lilac and mandarin leaves and leaf petioles and on mandarin twigs as is described by Bryan (1928).
It should be noted that the lack of molecular data in literature sources concerning the genetic identity and structure of
Results of MST showed that
Further detailed biological studies, using more strains of
This work was supported by the Ministry of Education and Science, Republic of Serbia (Grants No. TR31018) and the Ministry of Science, Montenegro (Grant “Invasive species”).