First Report of Xanthomonas campestris pv. campestris as the Causal Agent of Black Rot on Oilseed Rape (Brassica napus) in Serbia

Abstract

In September 2010, leaves of oilseed rape (Brassica napus L.) with v-shaped, necrotic lesions on the leaf margins surrounded by yellow halos were collected. Symptoms were observed on the domestic cultivar Slavica (IFVC, Novi Sad) located in the Bačka region, Vojvodina, Serbia, from a 3-ha field. Average disease incidence on 3-month-old plants was 45% (15 to 75%). Diseased leaves were rinsed in sterilized distilled water (SDW) and dried at room temperature for isolations. Leaf sections taken from the margin of necrotic leaf tissue were macerated in SDW and the extract was streaked onto yeast extract-dextrose-calcium carbonate (YDC) agar. Plates were incubated at 28°C for 3 days. Colonies were yellow, translucent, circular, and raised. Ten representative strains tested further were all gram-negative, catalase-positive, and oxidase-negative. The partial 16S rDNA sequence of a representative strain, TUr1, was amplified using primers fD1 and rD1 (2), and determined using the IMGGI SeqService facility in Belgrade. The 1,510-bp 16S rDNA sequence of TUr1 was compared to that of known strains in the NCBI GenBank database, and showed greatest similarity with that of Xanthomonas campestris pv. campestris (Xcc) strains ATCC 33913 and B100 (99% homology). Pathogenicity of 10 strains grown for 48 h on YDC at 28°C was completed using each of three methods: spraying a bacterial suspension (108 cfu/ml) onto the leaf surfaces of oilseed rape plants, stabbing the major veins of each of the first two true leaves with the tip of a sterile toothpick that had been dipped into a colony of the appropriate strain, and immersing cotyledons of the plants into a bacterial suspension (108 cfu/ml). All three tests were performed on 4-week-old oilseed rape plants of the cultivar Slavica. SDW was used for the negative control treatment for each method of inoculation. Reference strain Xcc NCPPB 1144 was used as a positive control treatment. Tests plants (two for each method of inoculation and each bacterial strain or control treatment) were maintained in a greenhouse at 25 ± 1°C and 80% relative humidity by keeping the plants in plastic bags. Two control plants for each of the negative and positive control treatments for each inoculation method were also enclosed in separate plastic bags. The bacterial strains and reference strain caused yellow lesions on inoculated plants that turned necrotic starting about 7 days after inoculation (DAI). The spots coalesced within 21 DAI to form necrotic areas. Plants inoculated with SDW remained symptomless. Reisolations were done onto YDC as described above. Reisolated strains showed the same colony morphology as described above. The bacterial strains grew at 35°C; produced levan from sucrose, hydrogen sulfide, and indole; did not reduce nitrate; hydrolyzed Tween 80; starch, gelatin, and aesculin; did not show tolerance to 0.10 and 0.02% triphenyl-tetrazolium chloride; and produced acid from d-arabinose, arginine, dulcitol, galactose, d-glucose, maltose, mannose, sorbitol, sucrose, and xylose (1). All strains tested by Plate Trapped Antigen-ELISAs (ADGEN Phytodiagnostics, Neogen Europe Ltd., Scotland) reacted with Xcc-specific polyclonal antibodies. Based on these tests, the strains were identified as Xcc. To our knowledge, this is the first report of this pathogen causing black rot of oilseed rape in Serbia. References: (1) T. B. Adhikariand and R. Basnyat. Eur. J. Plant Pathol. 105:303, 1999. (2) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

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

“…The results confirm the genetic heterogeneity among Xcc isolates that was established by Popovi c et al (2013a), where two major clusters of Serbian Xcc isolates from broccoli (first), kale (first) and cabbage (second) isolated in 2010 were revealed using GTG 5 and ERIC rep-PCR primers. Subsequently, ERIC and GTG 5 proved to be useful tools for fast estimation of pathovar diversity within 10 Serbian oilseed rape Xcc strains, also isolated in 2010, separating them into two (98% similarity) and three (92% similarity) groups, respectively (Popovi c et al, 2014), thereby anticipating the differences at the genetic level obtained in the present study with isolates obtained 4 years later. In the study of Singh et al (2011), rep-PCR (REP-, BOX-and ERIC-PCR) oligonucleotide primers also showed an ability to separate five different groups of Xcc isolates from different Brassica cropscauliflower, cabbage, broccoli, kohlrabi and turnipfrom the northern region of India, indicating the presence of genetic diversity within this X. campestris pathovar.…”

“…Host many cultivated cruciferous crops (Popovi c et al, 2013a(Popovi c et al, , 2014. In the light of its destructive potential, 147 isolates collected from seven brassicas (broccoli, cabbage, cauliflower, collard greens, kale, kohlrabi and winter oilseed rape) were subjected to examination of their genetic and pathogenic features in the present study.…”

“…In later years, Xcc started causing damage on other cultivated crucifers – cabbage, cauliflower and kale (Jovanović et al , ; Obradović & Arsenijević, ). The first appearance on winter oilseed rape ( B. napus ) was reported in 2010 (Popović et al , ).…”

“…Black rot of crucifers, caused by the plant pathogenic bacteria Xanthomonas campestris pv. campestris (Xcc), is a serious and destructive disease which is gradually spreading worldwide (Popovi c et al, 2013a(Popovi c et al, , 2013b(Popovi c et al, , 2014Singh et al, 2016). The pathogen is known to affect all cultivated brassica crops in Serbia that belong to two Brassica species, viz.…”

Terapia fotodinâmica e nanopartículas no controle de Xanthomonas campestris pv. campestris in vitro e em sementes de canola naturalmente contaminadas

“…campestris (Pammel) Dowson (Xcc), foi descrita pela primeira vez por Garman em 1890 (ALVAREZ, 2000). A partir de então, esta doença vem sendo relatada em todas as regiões do mundo onde há o plantio de brássicas (ALVAREZ;CHO, 1978;THAVEECHAI, 1983;MASSOMO et al, 2003;MIRIK et al, 2008;JENSEN et al, 2010;POPOVIĆ et al, 2013) e tem causado perdas de produtividade e de qualidade dos produtos (VILLETH et al, 2009).…”