Copper resistance in Xanthomonas campestris pv. campestris affecting crucifers in Trinidad

Fifty-six native isolates collected in 12 farming districts of Trinidad and seven reference strains of Xanthomonas campestris pv. campestris were evaluated for resistance to copper in buffered (pH 7.0) and unbuffered (pH 5.6) nutrient agar media. All isolates and reference strains were pathogenic and elicited typical black rot symptoms on a susceptible variety of Brassica olearceae, ‘Copenhagen Market’. Thirty-four and thirty-three native isolates were highly resistant to copper (growth on?≥?200 ppm copper) in buffered and unbuffered media, respectively; however, all the reference strains were highly susceptible to copper. The mean minimum inhibition concentration for the 56 native isolates was 224.6 ppm copper indicating that high levels of copper resistance are present in X. campestris pv. campestris in Trinidad. The association between growth of the 56 isolates and seven reference stains on buffered and unbuffered media was strong (Pearson’s and Spearman’s r?=?0.93; P?<?0.01) suggesting that either medium can be used to evaluate resistance to copper in X. campestris pv. campestris. There was also a strong association between length of time of continuous applications of copper formulations to treat black rot disease and proportion of the native X. campestris pv. campestris with resistance to copper (Pearson’s r?=?0.96; Spearman’s r?=?0.93); however, there was no association between resistance to copper and aggressiveness at 10 days after inoculation.     

Characterisation of Xanthomonas campestris pv. campestris isolates from South Africa using genomic DNA fingerprinting and pathogenicity tests

The genetic diversity of Xanthomonas campestris pv. campestris isolates from South Africa was evaluated using 28 isolates obtained from the Johannesburg Fresh Produce Market. Samples were collected from cabbage supplies from farms in Gauteng, Mpumalanga and North West Provinces. Strains were isolated from small sections of infected cabbage leaf samples and cultured on Yeast Dextrose Agar. Isolates identity was confirmed by ELISA and Pathogenicity test. Pathogenicity tests were performed by inoculating leaves of known susceptible cabbage seedlings. Infection symptoms induced could be categorized into three groups, ranging from typical to non-typical black rot symptoms. Four differential Brassica cultivars with known avirulence genes were used for race typing done by spray inoculation. Four races, namely 1, 3, 4 and 6, were identified. Of the 28 isolates, four were identified as race 1, two as race 3, 19 as race 4 and three as race 6. Repetitive DNA polymerase chain reaction-based fingerprinting using Eric- and Box-primers was used to assess the genetic diversity. Generated fingerprints of X. c pv. campestris were relatively similar. Cluster analysis could not strictly group isolates by their geographical origin, suggesting limited diversity of Xanthomonas campestris pv. campestris strains within cabbage producing regions in South Africa.     

Identification and Characterisation of Xanthomonas campestris pv. campestris Strains from Tanzania by Pathogenicity Tests, Biolog, rep-PCR and Fatty Acid Methyl Ester Analysis

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc), is a major disease constraint to cabbage production by smallholder farmers in Africa. Variability exists within the pathogen, and yet differentiation of Xcc strains from other closely-related xanthomonads attacking crucifers is often difficult. The Biolog system, fatty acid methyl ester analysis using microbial identification system (MIS), rep-PCR and pathogenicity tests were used to identify and characterise Xcc strains from Tanzania. Great diversity was observed among Xcc strains in their Biolog and rep-PCR profiles. Specific rep-PCR genomic fingerprints were linked to some geographical areas in the country. Most of the Xcc strains were clustered in two groups based on their fatty acid profiles and symptom expression in cabbage although some deviant strains were found. Each of the methods allowed a degree of identification from species, pathovar to the strain level. Biolog and MIS identified all Xcc strains at least to the genus level. Additionally, Biolog identified 47% of Xcc strains to the pathovar and 43% to strain level, whereas MIS identified 43% of the strains to pathovar level. In the absence of a database, the utility of rep-PCR for routine diagnosis of strains was limited, although the procedure was good for delineation of Xcc to the strain level. These findings indicate the existence of Xcc strains in Tanzania that are distinct from those included in Biolog and MIS databases. The limitations noticed warrant continued improvement of databases and inclusion of pathogenicity testing, using universally susceptible cultivars, as an integral part of strain identification.     

Growth of Xanthomonas campestris pv. campestris populations at constant and variable temperatures

Quantitative data were collected to describe the relation between temperature and growth of the cabbage black rot pathogen,Xanthomonas campestris pv.campestris (Xcc). Relative growth rates derived from experiments at constant temperatures were used in dynamic simulation of bacterial population development. The relative growth rates were adequate to simulate growth ofXcc populations at constant temperatures but overestimated growth of populations at variable temperatures. This finding gives rise to the hypothesis, that under field conditions, disease development is slower than is expected on the basis of growth parameters obtained from studies with constant temperatures.     

Field evaluation for resistance to the black rot pathogen <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis> in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis>)

Black rot, caused by Xanthomonas campestris pv. campestris, (Xcc), is one of the most serious diseases of crucifers world-wide. Forty-nine genotypes were evaluated for resistance under field conditions in Tanzania after artificial inoculation with Xcc race 1. Open pollinated white cabbage cultivars were generally susceptible, while Portuguese and pointed cabbages exhibited partial resistance. Some F1 white cabbage cultivars were highly susceptible, whereas others exhibited a high level of partial resistance. The most promising of the hybrid cultivars were T-689 F1, Gianty F1, No. 9690 F1, N 66 F1, and SWR-02 F1. Breeding line Badger I-16 exhibited the highest level of resistance of all genotypes. The genotypes accounted for 72.9–75.5% of the variation of the disease severity when assessed on the leaves, and 71.4% of the variation when assessed as internal black rot in heads at harvest. High correlations (equal to or above 0.7) were found between disease severities assessed on leaves three times during the growing season and also with the amount of internal black rot in heads. Leaf loss also was correlated with disease severity. The high genetic determination of the trait and the high correlations between disease assessments indicate that selection for resistance to black rot will be efficient when field screenings are carried out. Evaluation of genotypes for disease severity on leaves during the growing season combined with evaluations of head resistance in the most promising genotypes may be a simple method to select resistant cultivars.     

Growth of Xanthomonas campestris pv. campestris populations at constant and variable temperatures

Quantitative data were collected to describe the relation between temperature and growth of the cabbage black rot pathogen,Xanthomonas campestris pv.campestris (Xcc). Relative growth rates derived from experiments at constant temperatures were used in dynamic simulation of bacterial population development. The relative growth rates were adequate to simulate growth ofXcc populations at constant temperatures but overestimated growth of populations at variable temperatures. This finding gives rise to the hypothesis, that under field conditions, disease development is slower than is expected on the basis of growth parameters obtained from studies with constant temperatures.     

Survival of Xanthomonas campestris pv. campestris in the phyllosphere and rhizosphere of weeds

The phyllosphere and rhizosphere of weeds are important niches for phytobacterial survival. The absence of information in Brazil regarding Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot in crucifers, motivated this study. Twenty‐six weed species belonging to 14 botanical families were included in field experiments between August 2014 and October 2015. Lepidium virginicum and Raphanus raphanistrum (Brassicaceae) demonstrated great potential for survival of Xcc in the phyllosphere, with the bacterium isolated after 56 and 70 days, respectively. Low variation between maximum and minimum temperatures, high rainfall and high relative humidity at specific times of the year contributed to longer Xcc survival periods in the phyllosphere of some species. Xcc survived in the rhizosphere only in R. raphanistrum, where it was isolated for up to 28 days. No relation was found between climatic factors and survival in the rhizosphere. The data indicate that control of brassicaceous weeds will contribute to the control of black rot.     

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.     

Symptomless Spread of Blight-inducing Strains of Xanthomonas campestris pv. campestris on Cabbage Seedlings in Misted Seedbeds

Xanthomonas campestris pv. campestris induces two types of symptoms, namely, black rot and blight. Black rot symptoms are V-shaped lesions and black veins on the leaf, and blight symptoms are sudden collapse of interveinal tissues following the lack of veinal necrosis at early stages of infection. These two symptoms can occur simultaneously. However, the tendency to induce either symptom type is strain-dependent. Six strains were evaluated for their rate and pattern of spread in misted seedbeds by using strain-specific monoclonal antibodies and miniplate enrichment/ELISA. Data on pathogen incidence was defined as the presence of the pathogen in or on plants rather than visual symptoms. The results indicated that blight-inducing strains spread to more seedlings than black rot-inducing strains. The high incidences of blight-inducing strains in experimental plots were associated with non-randomness of spatial pattern of pathogen spread, indicating that high incidence is primarily due to the spread from adjacent plants by leaf contact and water splash. Most ELISA-positive seedlings were symptomless, indicating that the sensitivity of the system used in this study was adequate for detection of latent or epiphytic spread.     

A PCR-based assay for differentiating A- and A*-type strains of Xanthomonas citri subsp. citri, the causal agent of Asiatic citrus canker

By applying A- and A*-type strains of Xanthomonas citri subsp. citri (Xcc) in a repetitive sequence-based polymerase chain reaction (rep-PCR), two DNA amplicons, one unique to each strain, were evaluated as a probe against the DNA of Xcc strains. Two pairs of primers derived from these amplicons were tested in a PCR analysis. The results confirmed that primers Ms⁺/Ms^? are useful for differentiating A-type from A*-type strains of Xcc. Also, a multiplex PCR with both set of primers can be used to distinguish three groups in Xcc populations: A-type strains and two subgroups of A* strains including Iranian and Thai A* strains.     

Multilocus sequence typing analysis of Italian Xanthomonas campestris pv. campestris strains suggests the evolution of local endemic populations of the pathogen and does not correlate with race distribution

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in Brassicaceae. It is widespread in Italy and severe outbreaks occur under conditions that favour disease development. In this study a multilocus sequence typing approach (MLST) based on the partial sequence of seven loci was applied to a selection of strains representative of the main areas of cultivation and hosts. The aim was to investigate whether the long tradition of brassica crops in Italy has influenced the evolution of different Xcc populations. All loci were polymorphic; 14 allelic profiles were identified of which 13 were unique to Italian strains. Based on the seven loci, the most common genotype within the Italian Xcc strains (AP1) was also the most representative genotype found in worldwide Xcc strains. This genotype was included in a new clonal complex in addition to three other clonal complexes already identified in Xcc populations. The phylogenetic reconstruction using a concatenated dataset of four conserved protein-coding genes, dnaK, fuyA, gyrB and rpoD, showed that the Italian strains belonged to two genetic groups. Physiological races were also investigated for the first time in Italy. The race structure of Xcc was determined by inoculating eight differential Brassica lines belonging to five species and showed that, in Italy, race 4 is the most widespread, followed by races 1 and 6. No correlation was found between allelic profiles, host of isolation, geographical origin and races, although a prevalent race was identified within the same clonal complex.     

Improved control of black rot of broccoli caused by <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis> using a bacteriophage and a nonpathogenic <Emphasis Type="Italic">Xanthomonas</Emphasis> sp. strain

We examined the potential for biological control of black rot of broccoli, caused by Xanthomonas campestris pv. campestris (Xcc), using nonpathogenic Xanthomonas sp. strain 11-100-01 (npX) mixed with bacteriophage XcpSFC211 (pXS). Inoculation of intact broccoli plants in greenhouse trials with either npX or pXS did not control black rot. After injured plant inoculation, however, npX alone or npX with pXS significantly controlled black rot. When a mixed suspension of npX with pXS was placed on a membrane filter, then washed with distilled water and air-dried, a substantial amount of pXS adsorbed to the surface of npX. In a field trial, broccoli plants were sprayed with a suspension of npX with pXS, then inoculated with Xcc. A meta-analysis of the results from five field trials showed an integrated risk ratio (IRR, the ratio of disease incidence in inoculated broccoli plants to the incidence in control plants) of 0.69 after treatment with only npX and 0.59 with npX with pXS, indicating that black rot incidence was significantly reduced by each treatment. The difference between these two treatments was also significant. IRR was 1.24 when comparing suppression by npX with pXS and that by basic copper sulfate wettable powder; thus, their control was comparable. The combination of npX with pXS improved the preventive effect against black rot. This is the first report describing that a nonpathogenic Xanthomonas sp. strain mixed with a bacteriophage effectively controlled black rot of broccoli in field trials.     

Phenotypic and Genotypic Characterization of Xanthomonas campestris pv. zinniae Strains

During 1997 and 1998, serious outbreaks of bacterial leaf spot disease were observed on zinnia plants grown in home and commercial gardens in Ohio, USA. Twenty-two strains of Xanthomonas campestris pv. zinniae, isolated from diseased zinnia plants and contaminated seeds, were identified based on morphological, physiological and biochemical tests, fatty acid methyl ester analyses and pathogenicity tests on zinnia cv. Scarlet. Host range studies indicated that all of the X. campestris pv. zinniae strains were pathogenic on zinnia and tomato, but not on cabbage, lettuce, pepper and radish. The phenotypic and genotypic relationships among the strains determined based on serological reaction pattern, fatty acid profiles, repetitive extragenic palindromic-polymerase chain reaction (rep-PCR) fingerprints and sequence analysis of the 16S–23S rDNA spacer region suggested that X. campestris pv. zinniae strains were closely related to each other, but clearly distinct from other Xanthomonas species including X. campestris pv. campestris, X. axonopodis pv. vesicatoria, X. vesicatoria and X. hortorum pv. vitians tested in this study. The results also demonstrated that rep-PCR fingerprinting is rapid, reliable and the most practical method for routine detection and identification of X. campestris pv. zinniae strains.     

Genetic variability of Iranian strains of Pseudomonas syringae pv. syringae causing bacterial canker disease of stone fruits

Strains of Pseudomonas syringae pv. syringae (Pss) were isolated from healthy and diseased stone fruits tissues sampled from 38 stone fruits orchard sites in Iran in 2010 and 2011. These strains were tested for pathogenicity and the presence of the syrB gene and were genetically characterized by using ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and IS50 (insertion sequences) primers and PCR. All 78 strains of Pss tested were moderately to highly pathogenic on Loring peach seedlings. A total of 78 isolates of the Pss amplified a 752-bp fragment with the syrB primers. To assess genetic diversity among the strains, genomic DNA was extracted from strains and used in rep-PCR and IS50-PCR analysis. Cluster analysis was performed using UPGMA. The strains of Pss were separated into nine distinguishable genotypic groups by the combination data set of both rep-PCR and IS50-PCR at 73 % similarity level. There was no significant correlation between genetic diversity and geographical origin of the isolates. These results indicate that a combination of rep-PCR and IS50-PCR fingerprinting can be used as a high resolution genomic fingerprinting method for elucidating intrapathovar diversity among strains of Pss. The results of this study demonstrated the existence of a considerable genetic diversity among Pss strains causing canker of stone fruit trees in Iran. In this study, genetic variability in Iranian strains of Pss were established, which will be of immense use in the development of resistant genotypes against this bacterial pathogen.     

Characterization of isolates that cause black rot of crucifers in East Africa

A study was conducted in the East African countries of Kenya, Tanzania and Uganda in the months of July and August 2009 with the objectives of assessing the status of black rot and race structure of Xanthomonas campestris pv. campestris in the three countries. Samples infected with black rot were collected from farmers’ fields mainly from Brassica oleracea crops (broccoli, cabbage, cauliflower and kales). A total of 399 farms were surveyed of which 260 were from Kenya, 91 from Tanzania and 48 from Uganda. Following successful isolations, a total of 249 isolates of the causal agent, Xanthomonas campestris pv. campestris were recovered. Pathogenicity of all isolates was confirmed on B. oleracea susceptible cultivars Copenhagen Market F1 and Wirosa F1. Sixty of the 250 isolates were race-typed using a differential set Brassica spp. Only two races, 1 (Kenya and Tanzania) and 4 (Kenya, Tanzania and Uganda) were observed however, another race (5) was observed from one isolate recovered from a B. rapa sample obtained from Tanzania in 2003. Genomic fingerprinting with repetitive-PCR revealed clusters that did not depict significant correlations between isolates and geographical location, isolates and host adaptation or isolates and race. However, it did demonstrate existence of genetic differences within the East African X. campestris pv. campestris population indicating that it is not a similar clonal population of the same genetic background.     

Variation in Genotype and Aggressiveness of Ralstonia solanacearum Race 1 Isolated from Tomato in Taiwan

ABSTRACT A population of Ralstonia solanacearum race 1 from tomato (Lycopersicon esculentum) was analyzed for genetic polymorphism and aggressiveness on tomato. The 46 strains were collected from main tomato-growing areas in Taiwan. Genetic analysis was achieved by two polymerase chain reaction (PCR)-based methods: REP-, ERIC-, and BOX-PCR (collectively as rep-PCR) and random amplified polymorphic DNA (RAPD) techniques. RAPD (with three 10-mers) and rep-PCR revealed 35 and 30 haplotypes, respectively, that were grouped in 14 clusters and 3 clusters, respectively. Distribution of strains into genetic clusters did not appear related to biovar or geographic origin in considering RAPD, rep-PCR, or composite data. Although strains were more dissimilar based on RAPD data than on rep-PCR data, the two techniques gave complementary results for strain clustering. A set of 40 strains representing the main haplotypes was inoculated on six tomato cultivars differing in their bacterial wilt resistance. Six groups differing in general level of aggressiveness and cultivar specificity were detected. Although populations were highly diverse in both genotype and aggressiveness, no association was found between the two characteristics. Although the sample sizes in this study were not adequate to draw definite conclusions about population structure, these results will be valuable for future population genetic studies on R. solanacearum.     

Biological Control of Black Rot (Xanthomonas Campestris Pv. campestris) of Brassicas with an Antagonistic Strain of Bacillus Subtilis in Zimbabwe

Biological control efficiency of an antagonistic, endophytic strain of Bacillus subtilis (strain BB) was evaluated against three strains of the black rot pathogen, Xanthomonas campestris pv. campestris (Xcc), in four Brassica crops (cabbage, cauliflower, rape and broccoli) grown during three consecutive growing seasons and on two soil types, in two different areas in Zimbabwe. Strain BB controlled the disease caused by strain Xcc B-147 in all Brassica crops during the dry and short rainy seasons. A similar effect was observed in cabbage using the strain Xcc 33908. Biological control was effective in broccoli, but not in cabbage and rape during the main rainy season in clay loam soil and limited biological control effect was still observed when these crops were grown in sandy loam soil. The endophytic colonisation of cabbage roots by strain BB was confirmed by immuno-blotting during the whole growing season. Biological control of black rot with strain BB is discussed in relation to its effect on Xcc strains, Brassica crops and to the effect of weather and soil conditions.     

Characterization and differentiation of soft rot causing Pectobacterium carotovorum of Indian origin

Twenty different strains of Pectobacterium carotovorum (Pcc) were recovered from vegetable-growing fields of Vadodara, Gujarat (India) using a plant host enrichment approach during the years 2006–9. The isolated strains, based on differences in physiological and biochemical features, were classified into five different biovars, and then identified as Pectobacterium carotovorum (Pcc) by species-specific PCR and 16S rDNA sequences. Moreover, these Pcc strains were also differentiated based on virulence traits, plant cell wall degrading enzymes production and phylogenetic analysis of 16S rDNA sequence and Repetitive extragenic palindromic—PCR (rep-PCR). High genetic variability, independent of their pathogenicity, was revealed among these Pcc strains by rep-PCR typing using four different primer sets . Factors other than the plant host specificity seem to correlate with genetic variability of these economically important Pcc strains. Significantly, polyphasic characterization of the Pcc strains clearly reveals the heterogeneity among them. The present studies can be considered as useful epidemiological surveillance (or distribution) of soft rot causing Pcc in the semi arid region of India.     

Molecular characterization,comparison of screening methods,and evaluation of cross‐pathogenicity of black rot (Xanthomonas campestris pv. campestris) strains from cabbage,choy sum,leafy mustard and pak choi from Taiwan

Choy sum (Brassica rapa var. parachinensis), leafy mustard (Brassica juncea) and pak choi (B. rapa var. chinensis) are highly nutritious components of diets in Taiwan and other Asian countries, and bacterial black rot caused by Xanthomonas campestris pv. campestris (Xcc) is a major biotic constraint in these crops. As very little was known about the Xcc strains from these crops in these regions, including their cross‐pathogenicity and aggressiveness on different hosts, Xcc strains were obtained from cabbage (Brassica oleracea var. capitata), choy sum, leafy mustard and pak choi crops in Taiwan. Two previously published PCR‐based assays reliably distinguished the Xcc strains from other Xanthomonas species and subspecies. Phylogenetic analysis based on repetitive sequence‐based PCR assays placed the Xcc strains in a clade distinct from other Xanthomonas species, and also showed host specificity. Although all of the Xcc strains from the different host species were pathogenic on all five Brassica test species in both a detached leaf assay and an intact plant assay, in the intact plant assay they showed differences in virulence or aggression on the different test hosts. The Xcc strains from leafy mustard and pak choi were consistently highly aggressive on all the test host genotypes, but the strains from choy sum and cabbage were less aggressive on leafy mustard and choy sum. The intact plant assay proved more discriminating and reliable than the detached leaf assay for comparing the aggressiveness of Xcc strains on different host genotypes, and so, with the new Xcc strains isolated in this study, will be useful for screening leafy brassica germplasm accessions for resistance to black rot.     

Discrimination of <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis> races among strains from northwestern Spain by <Emphasis Type="Italic">Brassica</Emphasis> spp. genotypes and rep-PCR

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc) is a severe seedborne disease of Brassica crops around the world. Nine races are recognized, being races 1 and 4 the most aggressive and widespread. The identification of Xcc races affecting Brassica crops in a target area is necessary to establish adequate control measures and breeding strategies. The objectives of this study were to isolate and identify Xcc strains from northwestern Spain by using semi-selective medium and pathogenicity tests, determine the existing races of Xcc in this area by differential series of Brassica spp., and evaluate the use of repetitive DNA polymerase chain reaction-based fingerprinting (rep-PCR) to differentiate among the nine existing Xcc races. Seventy five isolates recovered from infected fields were identified as Xcc. Race-typing tests determined the presence of the following seven pathogen races: 1, 4, 5, 6, 7, 8 and 9. Race 4 was the most frequent in Brassica oleracea and race 6 in Brassica rapa crops, therefore breeding should be focussed in obtaining resistant varieties to both races. Cluster analysis derived from the combined fingerprints showed four groups, but no clear relationship to race, crop or geographical origin was found. Rep-PCR analysis was found not to be a reliable method to discriminate among Xcc races, therefore race typing of Xcc isolates should be done by using the differential series of Brassica spp. genotypes or another alternative approach.