SURVIVAL OF CURTOBACTERIUM FLACCUMFACIENS pv ... - SIPaV

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Journal of Plant Pathology (2012), 94 (2), 331-337

Edizioni ETS Pisa, 2012

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SURVIVAL OF CURTOBACTERIUM FLACCUMFACIENS pv. FLACCUMFACIENS IN SOIL AND BEAN CROP DEBRIS T.A.F. Silva Júnior, D.R. Negrão, A.T. Itako, J.M. Soman and A.C. Maringoni Universidade Estadual Paulista Júlio de Mesquita Filho, Faculdade de Ciências Agronômicas, Departamento de Produção Vegetal, Caixa Postal 237, 18.603-970, Botucatu, São Paulo, Brazil

SUMMARY

The survival of Curtobacterium flaccumfaciens pv. flaccumfaciens was evaluated as free cells in the soil, under laboratory conditions, and on infected bean residues retained on the soil surface or buried 20 cm deep, in 2008 and 2009. Free bacterial cells survived in the soil between two and 16 days. Survival was influenced by soil type, moisture and incubation temperature of the samples. In field tests, climatic conditions and incorporation of infected crop residues influenced bacterial survival the most. C. flaccumfaciens pv. flaccumfaciens survived up to 240 days on bean residues retained on the soil surface, but 30 days only when these were incorporated 20 cm deep into the soil. Because C. flaccumfaciens pv. flaccumfaciens-infected bean debris can be an important source of inoculum, soil incorporation of crop residues and rotation using non host crops for the pathogen are recommended to prevent disease epidemics. Key words: bacteria, bean bacterial wilt, Phaseolus vulgaris, epidemiology.

Common bean (Phaseolus vulgaris) is a legume with high nutritional value consumed by millions of people around the world, serving as the primary source of protein in the diet of many developing countries such as Brazil (Broughton et al., 2003; Graham et al., 2003). Several diseases cause significant losses to bean production, among which those caused bacterial pathogens such as Xanthomonas axonopodis pv. phaseoli (Smith) Dye, Pseudomonas savastanoi pv. phaseolicola (Burkholder) Gardan et al., P. syringae pv. syringae van Hall, P. syringae pv. tabaci (Wolf and Foster) Young et al., and Curtobacterium flaccumfaciens pv. flaccumfaciens (Hedges) Collins and Jones (Cff), the causal agent of bacterial wilt (Saettler, 1991). Cff was first reported by Hedges (1922) in South Dakota (USA), where it caused yield losses of about 90% to bean crops, then in other Corresponding author: T.A.F. Silva Júnior Fax: + 55.14.3811.7206 E-mail: [email protected]

US states (Michigan, Virginia, Maryland, Montana and the District of Columbia) and Europe (Germany and France) (Hedges, 1926). In subsequent years, records came from Canada, Mexico, South America, Australia, Mauritius, Kenia and Tunisia. In Romania and Russia Cff has a restricted distribution, the same as in Albania, Greece, Hungary, Italy, Belgium, Bulgaria, Poland, Spain, Ukraine and Turkey, where it did not become established and caused minor economical losses (CABI, 2010). In Brazil, Cff was first reported in the State of São Paulo in 1995 (Maringoni and Rosa, 1997), and is currently present in other Brazilian states (Leite Jr. et al., 2001; Uesugi et al., 2003; Deuner et al., 2006; Maringoni and Theodoro, 2006; Theodoro et al., 2010). Phytopathogenic bacteria often survive by associating with seeds, infected plant organs, crop residues (in the soil as local residents) and epi- or endophytically in crops and/or weeds (Coyne and Schuster, 1974). Their survival is affected by multiple factors, i.e. temperature, humidity, pH, aeration, chemical, physical and biological characteristics of the soil. In general, the optimum temperature for survival in the environment varies between 20 and 37ºC (De Boer, 1982). Bean pathogenic bacteria, including Cff, do not survive outside plant tissues for a long time (Coyne and Schuster, 1977). They rather persist from one growing season to the next in the form of aggregates inside host tissues or on the plant surface as phylloplane residents. There are reports of Cff surviving in dried bean pods in herbariums for up to five years (Bulkholder, 1930), and in association with bean seeds in laboratory conditions for up to 24 years (Burkholder, 1945). The lack of information on the natural niches for Cff survival in nature, prompted the present study which aimed at: (i) assessing Cff survival in the form of free cells in two different soil types and under different temperatures and humidity levels in association with bean residues; (ii) ascertaining the influence of the incorporation of crop residues and that of different environmental conditions on bacterial survival.

MATERIALS AND METHODS

Bacterial isolates and pathogenicity. Seven mutant


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isolates of Cff resistant to 100 µg ml-1 rifampicin (Saettler and Weller, 1978) were obtained from isolate Feij2634 from the collection of the FCA/UNESP Laboratory of Phytobacteriology (Botucatu, São Paulo, Brazil) Nutrient agar (Schaad et al., 2001) containing 5 g l-1 sucrose (NSA) and 100 µg ml-1 rifampicin was used to obtain the mutants. Isolates were preserved in test tubes containing NSA plus 100 µg ml-1 rifampicin and sterilized mineral oil, and by lyophilization. A pathogenicity test was conducted using Cff-susceptible bean plants of cv. Pérola grown in pots with approximately 2 litres of substrate consisting of autoclaved soil, weathered cattle manure, and washed coarse sand (1:1:1), plus 0.6 kg ammonium sulfate, 1.7 kg superphosphate, 0.6 kg potassium chloride and 0.8 kg lime for every m3 of the mixture. The same substrate formulation was also used in the other tests. Three seedlings were grown in each pot which, 10 days after emergence, were inoculated by wounding the stem with an entomological needle moistened with colonies of the Cff mutants (Maringoni, 2002). Control seedlings were pricked with a needle dipped in sterile distilled water. Five groups of three seedlings were inoculated with each bacterial isolate, totalling 15 inoculated plants per isolate, which were kept under greenhouse conditions at temperatures ranging between 22 and 30°C. Disease severity was assessed 21 days after inoculation by assigning scores ranging from 0 to 9 (Maringoni, 2002). Mutant isolate Feij. 2634-4 was the most aggressive and was selected for the survival experiments. Survival of C. flaccumfaciens pv. flaccumfaciens as free cells in the soil. In this experiment two types of soil were used: soil 1, a Red Nitossol with clayey texture (organic matter 26 g dm-3, pH 4.3); and soil 2, a Claysoil, medium texture (organic matter 15 g dm-3, pH 4.1). The two soils were sifted separately through a mesh sieve 8 (2.8 mm opening), dried in a greenhouse for five days and used to fill 200 ml polystyrene cups with 150 g of each soil type. To test for soil infestation, isolate Feij.2634-4 was sown on NSA and incubated at 28°C for 72 h. After incubation, bacterial suspension was adjusted using colorimetry (A540nm = 0.1) to 108 CFU ml-1. The cups containing soil samples received an amount of bacterial suspension and distilled water sufficient to raise their moisture contents to 50 and 100% field capacity. To achieve 50% field capacity, the cups received 35 ml of bacterial suspension, whereas for 100% field capacity, 35 ml of bacterial suspension plus 35 ml of distilled water were administered. After infestation, cups were sealed with aluminum foil, weighed and placed in bacteriological incubators at 20, 25 and 30°C. Every three days all cups were weighed and the evaporated water was replaced. One hundred twenty samples for each treatment were analysed to ascertain Cff survival


in the soil for 30 days. Every day, the content of four cups incubated at the three above temperatures was individually transferred to previously autoclaved 2,000 ml beakers, totaling three 600 g soil samples. These samples were homogenized individually with a sterilized glass rod and aliquots (25 g) from each sample were transferred to a 1,000 ml flask containing 250 ml sterile distilled water. The flasks were shaker-agitated at 200 rpm for 1 h at 25°C, let to sediment for 30 min, the supernatant was removed and diluted (100 to 10-4), then 100 µl from undiluted and diluted suspensions were spiral-plated with a sterile drigalski stick onto a modified semi-selective culture medium for Cff (SSMCFF; Maringoni, 2006), consisting of NSA with the following compounds added aseptically after autoclaving per liter: Congo red (0.05 g), skimmed milk powder (5 g), chlorothalonil (0.01 g), methyl thiophanate (0.01 g) and rifampin (0.01 g). Plates were incubated at 28°C for 72 h. Colonies with characteristics similar to those of Feij.2634-4 mutant were quantified, and the data were log10 transformed and expressed as CFU g-1 soil. In each treatment, six typical Cff colonies were selected, and subjected to differential Gram stain, growth in NSA containing 7% NaCl, pathogenicity on bean plants cv. Pérola and biochemical characterization using Biolog/ Microlog (Câmara et al., 2009). Survival of C. flaccumfaciens pv. flaccumfaciens in association with bean residues under field conditions. Three experiments were carried out in the Lageado Experimental Farm, College of Agriculture, Botucatu, State of Sao Paulo (22°50’40” S; 48°26’08” O) on a field with clay soil, medium texture, from February 2008 to November 2008 (trial I), November 2008 to June 2009 (trial II) and July 2009 to January 2010 (trial III). Potted bean seedlings cv. Pérola were inoculated ten days after emergence (Maringoni, 2002) with isolate Feij.2634-4. Thirty days after inoculation, plants were collected and placed for seven days in a forced air laboratory oven at 30°C. Three entire plants were then transferred to nylon bags measuring 20 x 30 cm (Casa et al., 2003). The bags were closed with a plastic ribbon and left on the soil surface, or buried 20 cm deep in the soil. Four replicates (R1, R2, R3 and R4) with sufficient material for the whole duration of the experiment (12 months) were prepared. Every 15 days, four surface and four buried bags were randomly removed, their contents were weighed and crushed in sterilized phosphate buffered saline (PBS) 0.01 M, pH 7.0. To each gram of crushed plant material 10 ml of sterile PBS 0.01 M was added. The mixture was triturated using a Turrax TE-102 (Turratec, Brazil). The suspension was serial diluted (100 to 10-4) onto the modified semi-selective SSMCFF (Maringoni, 2006) and incubated at 28°C for 96 h. From each sample, typical colonies similar to those of


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Feij. 2634-4 were purified and subjected to a differential Gram stain, growth on NSA containing 7% NaCl, pathogenicity on bean plants cv. Pérola and characterization by the Biolog/Microlog method. A qualitative assessment of the presence of Cff was also done for the samples collected (Torres et al., 2009). A sample was considered a carrier of Cff when at least one bacterial colony could be isolated from it. When bacteria were not isolated from at least two subsequent samples (30 days), bacterial survival was determined as the period up to the last sampling containing bacterial growth. Climatic data (minimum, medium and maximum temperatures and precipitation) for the period of the experiments were obtained from a meteorological station, located approximately 300 m from the assay.

RESULTS

Survival of C. flaccumfaciens pv. flaccumfaciens in the soil as free cells. Cff survival in the soil as free cells was influenced by the incubation temperature of the samples, type and humidity of the soil. The bacterium survived for a period of up to 16 days in soil 1, and up to 9 days in soil 2 (Table 1). Regardless of soil type and moisture content, survival was longest at 20°C, intermediate at 25°C, and shortest at 30°C. As for the moisture content, bacteria lived longer when the soil was maintained at 50% field capacity: between 8 and 16 days for soil 1, and between 5 and 9 days for soil 2 (Table 1). Survival of C. flaccumfaciens pv. flaccumfaciens in association with bean residues under field conditions. The duration of Cff survival in infected bean residues retained on the soil surface or buried 20 cm deep, is

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shown in Table 2. The bacterium survived 30 days in trials I and III, and less than 15 days in trial II, in residues buried 20 cm deep. In samples maintained at the soil surface, the bacterium survived for 240 days in trial I, 195 days in trial II and 165 days in trial III. Climatic data during the course of the three trials are shown in Fig. 1, 2 and 3. During the first 15 days after the beginning of the trials, precipitation levels were 48.2 mm in trial I, 40.8 mm in trial II, and 3.8 mm in trial III. In the first 30 days of trials I and III, precipitation levels were 176.9 mm and 91.9 mm, respectively. In the first 165 days after the beginning of the trials, the level of precipitation during trial I was 358 mm, 849.9 mm during trial II and 1.012 mm during trial III. Comparing the first 195 days of trials I and II, the rainfall was 459 mm during I and 871.5 mm during II. The maximum, minimum and average temperatures in the first 15 days of the trials were 28.0°C and 19.2°C during trial I, 29.0°C and 16.4°C during trial II, and 24.6°C and 13.8°C during trial III. In the first 30 days of trials I and III, these temperatures were 26.7°C and 18.3°C, and 23.7°C and 13.7°C, respectively. The average maximum and minimum temperatures during the first 165 days of the three trials were 25.1°C and 15.6°C during trial I, 27.6°C and 17.8°C during trial II and 26.6°C and 16.8°C during trial III. For the first 195 days of trials I and II, these temperatures were 25.2°C and 15.5°C and 27.0°C and 17.1°C, respectively.

DISCUSSION

Cff survival as free cells in the soil was relatively low. It was influenced by soil type, moisture content, and sample incubation temperature. When in the soil, Cff can be considered a “transient resident” because its

Fig. 1. Precipitation (mm), maximum, medium and minimum temperatures (°C), and periods when bean residues were collected in the field for assay I.


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cells do not last long enough to contribute to their own perpetuation (Buddenhagen, 1965). There are two reasons why most plant pathogenic bacteria, including Cff, have a short survival timing in the soil: they do not produce resistant spore and are inhibited by antagonists and predators (actinomycetes, other bacteria, fungi, protozoa and nematodes). Antibiotics produced by the antagonists were shown to be the main factor inhibiting the development of high populations of plant pathogenic bacteria (Buddenhagen, 1965). The addition of organic matter to the soil tends to increase the number of antagonistic microorganisms, further accelerating the reduction of bacterial populations (Patrick, 1954; Brian, 1957; Coyne and Schuster, 1974). The Cff population in the soils tested in this study was reduced more rapidly in samples stored at 100% field capacity and incubated


at higher temperatures, possibly because of an increased activity of microbial antagonists under these conditions. Even though the initial population of Cff was denser in soil 2 than in soil 1 on the day when the experiments were set up, bacterial survival was shorter in soil 2. Soil composition can influence the survival of many bacteria according to Hattori (1973), who observed that under conditions of low humidity bacteria survive longer within clusters of clay than on the soil surface and attributed this behaviour to increased availability of water within clay clots. This suggests that bacteria have a lower survival capacity in sandy soils with few aggregate formations than in more heavily textured soils such as clay. Clay aggregates form an envelope around the bacterial cells, resulting from electrostatic attraction between charged groups on the clay molecules and the bacterial

Fig. 2. Precipitation (mm), maximum, medium and minimum temperatures (°C), and periods when bean residues were collected in the field for assay II.

Fig. 3. Precipitation (mm), maximum, medium and minimum temperatures (°C), and periods when bean residues were collected in the field for assay III.


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Table 1. Survival period (days) of Curtobacterium flaccumfaciens pv. flaccumfaciens in two types of soil, two humidity levels, and three incubation temperatures.

Type of soil

Soil 1 Soil 2

Survival of C. flaccumfaciens pv. flaccumfaciens in soil (days) Humidity (50% of field capacity) Humidity (100% of field capacity) Temperature (°C) Temperature (°C) 20 25 30 20 25 30 16 11 8 14 7 5 9 6 5 7 3 2

cells. This wrap can protect bacterial cells during periods of desiccation and rehydration of the soil (Marshall, 1971). Messiha (2006) found that populations of Ralstonia solanacearum race 3 biovar 2 declined faster in sandy than in clayey soils, one reason being that sandy soils have a variety of species (biodiversity) lower than that of clayey soils. In trials on the persistence of Cff associated with bean residues, the survival period of bacteria in samples kept on the soil surface ranged from 165 to 240 days (Table 2). The analysis of climatic data collected during such tests showed that rainfall influenced bacterial survival (Fig. 1, 2 and 3). The intense precipitation during test III was associated with the shortest bacterial survival period, which may result from a faster degradation of bean residues retained on the soil surface. The water content, directly and/or indirectly, affects the viability of plant pathogenic bacteria within infected plant debris in the soil. In tropical and subtropical regions, the highest rate of decomposition of crop residues is associated with an intensive activity of many soil bacteria tha tutilize decomposing plant matter as an energy source (Leben, 1981; Chavez and Granada, 1988). The differences in temperature among the three trials of this study were very small and possibly had little influence on the survival period of Cff inside bean debris. In infected bean residues incorporated 20 cm deep into the soil, Cff survived for a period of less than 15 days to 30 days (Table 2). Although precipitation and temperature were similar during the first 15 days of trials I and II (Fig. 1 and 2), the bacterium survived for less than 15 days in trial II and up to 30 days in trials I and III.

Table 2. Survival periods of C. flaccumfaciens pv. flaccumfaciens on bean residues, maintained on the soil surface and buried 20 cm deep, in three different assays. Assay I II III

Survival of C. flaccumfaciens pv. flaccumfaciens (days) Soil surface 20 cm depth 240 30 195