Reaction of common bean lines to Xanthomonas axonopodis pv . phaseoli and Curtobacterium flaccumfaciens pv . flaccumfaciens

The aim of this study was to evaluate the resistance of 58 common bean lines against common bacterial blight (Xanthomonas axonopodis pv. phaseoli) and bacterial wilt (Curtobacterium flaccumfaciens pv. flaccumfaciens). The experimental design consisted of completely randomized blocks, with four replications per pathogen. The results were subjected to variance analysis by the F test at 1% probability. Significant differences between the treatments indicated different resistance levels among the lines against both pathogens. According to the Scott-Knott test, six lines were resistant to Xanthomonas axonopodis pv. phaseoli, 14 moderately resistant, and 38 susceptible. To Curtobacterium flaccumfaciens pv. flaccumfaciens, 11 lines were resistant, 26 moderately resistant and 21 susceptible. Among these, the lines Pr10-3-4/1, Pr10-5-2/1 and Pr10-52/2 of the black bean group and C10-2-4/2 of the Carioca group were resistant to both major bacterial diseases affecting common bean in Brazil.


INTRODUCTION
Common bean (Phaseolus vulgaris L.) is a major source of vegetable protein for direct human consumption.In addition, it also contains carbohydrates, dietary fiber, B-complex vitamins, iron, calcium, and other minerals, playing an important role in the diet of the Brazilian population (Vieira et al. 2006).
According to data of CONAB (2015), the mean grain yield in Brazil is about 1.095 kg ha -1 , well below the productive potential of a crop which, under appropriate conditions, can yield more than 4.000 kg ha -1 .This low productivity can be attributed to the incidence of pests and diseases, adverse environmental conditions, low-yielding cultivars, and sowing outside the agricultural zones (Oliveira et al. 2005).
Among the main diseases affecting common bean are common bacterial blight, caused by Xanthomonas axonopodis pv.phaseoli (Smith) Dye and bacterial wilt, caused by Curtobacterium flaccumfaciens pv.flaccumfaciens (Hedges) Collins & Jones.These pathogens are widespread in the producing regions, causing yield losses, especially when stimulated by favorable environmental conditions such as high temperatures (Theodoro 2004) The first pathogen symptoms of common bacterial blight appear on the shoot, consisting primarily of small watersoaked areas in the leaves, evolving to necrosis and imperfections in the seeds such as discoloration of the hilum, yellow spots, and wrinkling of the seed coat, which can reduce yields by 10 to 70% (Diaz et al. 2001, Bianchini et al. 2005).The inheritance of resistance to this pathogen is genetically complex, described by several authors as oligogenic or polygenic (Kelly et al. 2003, Santos et al. 2003, Manzanera et al. 2005).According to Zapata et al. (2010), Ferreira andGrattapaglia (2003), andMarquez et al. (2007), the number of genes, degrees and interactions involved in the expression of this trait may vary.Thus, the strong environmental influence in the evaluation period of the genotypes, can explain the low heritability observed in studies focused on the introgression of resistance into segregating common bean populations.Another factor that hampers the development of resistant genotypes for breeding programs is the genetic diversity of the pathogen (Mkandawire et al. 2004).
The pathogen symptoms of bacterial wilt begin with the colonization of vascular tissues, leading to the drying of apical leaflets, yellowing and gradual wilting of leaves, yellowish areas and necrosis of the parenchyma, as well as to yield drop (Maringoni 2002).According to Valentini et al. (2011), the resistance inheritance of this pathogen is polygenic and, according to Souza et al. (2006b), Wendland et al. (2008), and Torres et al. (2009b), the occurrence of genetic diversity and widespread dissemination in the producing regions of Brazil, makes the development of resistance sources even more difficult.For this reason, techniques have been developed to identify this bacterium in common bean crops (Maringoni 2002, Hsieh et al. 2005, Herbes et al. 2008), as well as to evaluate the resistance of genotypes and lines, with a view to the development of new resistant cultivars (Maringoni 2002, Souza et al. 2006a, Theodoro et al. 2007).
Both bacterial diseases are controllable by phytotechnical treatments such as crop rotation, elimination of crop residues and sowing of healthy seeds, whereas the use of resistant cultivars is the most efficient method to minimize production costs, avoiding significant yield and grain quality losses (Hsieh et al. 2005, Souza et al. 2006a, Huang et al. 2007b).

MATERIAL AND METHODS
The experiments were conducted at the Experimental Center of Farm Santa Elisa, Instituto Agronômico-IAC, in Campinas-SP (lat 22º 54' S and long 47º 03' W, and alt 854 m asl), from January 02 to June 30, 2014.The experimental design was arranged in completely randomized blocks, with four replications for each evaluated pathogen.Each repetition consisted of one pot with two plants.Inside the greenhouse, the temperature varied from 28 °C to 32 °C during the experimental period.
To evaluate the reactions of the lines to X. axonopodis pv.phaseoli (Common bacterial blight), seeds of 58 lines and the susceptible control (Rosinha G2) were disinfected with 70% ethanol and then with 1.25% sodium hypochlorite for 5 min.Subsequently, they were spread on paper sheets for germination and placed in BOD at 28 ºC for three days.After this period, the seedlings were transplanted into pots containing 500 g substrate (organic compound and soil, 1:1) and placed in the greenhouse.

T Ribeiro et al.
The isolate 11.280 of X. axonopodis pv.phaseoli, from the Plant Health Center of the Instituto Agronômico-IAC, Campinas, SP, was used.The isolate was multiplied on PDA (potato, dextrose, agar) and incubated at 28°C for 48 hours.Thereafter, inoculum was prepared by addition of distilled water and sterilization in the bacterial colony, scraping with a glass slide, and concentration adjustment to 108 CFU mL -1 .
Plants in the V 2 stage were inoculated by the technique of multiple needles, according to Pompeu and Crowder (1973).The primary leaves were perforated with light pressure to allow the pathogen to enter the plant.Then the pots were placed in a moist chamber for 48 hours, at temperatures between 25 °C and 28 °C, and then transferred to the greenhouse.
Ten days after inoculation, the plants were evaluated on a 1 -9 scale as follows: 1 to 2 -plants free of disease symptoms; 3 to 6 -small water-soaked areas; and 7 to 9 -plant tissue necrosis (Rava and Sartorato (1994).The resistance of genotypes was determined as follows: resistant lines had mean scores between 1 and 2; moderately resistant, between 2.1 and 5; and susceptible, between 5.1 and 9.
To evaluate reactions to C. flaccumfaciens pv.flaccumfaciens, seeds of the 58 lines and the pathogen-susceptible control (Rosinha G 2 ) were pre-germinated under laboratory conditions, as described above and transplanted into pots in the greenhouse.
The isolate used in this study was Feij-14627, provided by the Faculdade de Ciências Agronômicas, UNESP, in Botucatu.The isolate was multiplied in NA (Nutrient Sucrose Agar) culture medium and incubated at 28 °C for 72 hours.Inoculation was carried out in a greenhouse when the plants reached the V 3 developmental stage, by drilling two holes into the stem between the cotyledons and the primary leaves, using an entomological needle after dipping into the bacterial colony (Maringoni 2002).
The experiments were conducted separately.The results were subjected to analysis of variance (ANOVA) using the statistical software Genes (Cruz 2013), and differences between means were compared by the Scott-Knott test at 5% probability.

RESULTS AND DISCUSSION
The results of this study show the importance of knowing the resistance reaction of common bean genotypes to common bacterial blight and bacterial wilt.The evaluation of these genotypes is useful in breeding programs, to choose continuous sources of disease resistance, coupled with important agronomic traits, such as early cycle, high yield, upright growth, and resistance to grain darkening, with a view to develop superior genotypes for the productive sector.
The data of the evaluations of the 58 common bean lines regarding resistance to Xanthomonas axonopodis pv.phaseoli and C. flaccumfaciens pv.flaccumfaciens were subjected to analysis of variance by the F test at 1% probability.shows significant differences between treatments, indicating different resistance levels of the common bean lines to the two studied pathogens.The experimental precision, with coefficients of variation of 7.40% and 13.65%, indicated low environmental influence during the experiments, ensuring reliability of the results.
The low percentage of resistant genotypes to common bacterial blight can be explained by the occurrence of additive and non-additive effects, resulting in complex inheritance, as reported by Marquez et al. (2007).Six QTLs in F 3 plants resulting from the BAC-6 and HAB-52 cross were identified by Santos et al. (2003).Five of these QTLs were associated with resistance of leaves and one of pods, with a phenotypic variation from 12.7 to 68.7% for leaf and 12.9% for pod resistance.These results highlight the complexity of the trait, where the genes that control leaf resistance are not the same as those that control pod resistance, indicating the occurrence of oligo-or polygenic interaction, reinforcing the complex nature of resistance to the pathogen.
Among 56 evaluated cultivars, Silva (2009) identified 21 as resistant to common bacterial blight and among 61
According to Souza and Maringoni (2008), resistant genotypes involve the pathogen by protoplasmic projections, preventing its installation in the xylem vessels, while in susceptible genotypes the water transport is obstructed by the presence of bacterial cells.These results are related to the disease symptoms, e.g., plant wilting, yellowing, underdevelopment, and death, observed at different levels of aggressiveness in the 58 lines evaluated in this study.
The lines C10-2-4/2, Pr10-3-4/1, Pr10-5-2/1, and Pr10-5-2/2 (Table 3) were resistant to common bacterial blight and bacterial wilt, the two major bacterial diseases affecting common bean in Brazil.The development of these cultivars is extremely important to maintain the yield and grain quality of common bean, given the lack of resistant cultivars in the productive sector, the physiological variability and wide dissemination of the pathogens in crop areas.

Table 1 .
Summary of analysis of variance of 58 common bean lines inoculated with common bacterial blight (Xanthomonas axonopodis pv.phaseoli) and bacterial wilt (Curtobacterium flaccumfaciens pv.flaccumfaciens) ** Significant at 1% probability by the F test; CBB= Common Bacterial Blight and BW= Bacterial Wilt.