M 1 2 3 4 5 6

280bp**

M: DNA see marker (bp) PhiX 174 Hinf digest, Laae 1-4: DNA from soil contaminated with Ä sokmaetenm Biovar III, Larue 6: Positive control (pure DNA of £ solanacearum Biovar III), Larue 7: Negative control (DNA ofSoil free from Ä solanacearum).

Rs ForwardPrimerl: 5'-gTC gCC gTC AAC TCA CTT TCC-3'; Rs Reverse Primer 2: 5'-gTC gCC gTC AgC A AT gCg gAA TCg-3' (Opina et al 1997)

** Amplification of280bp DNA sequence indicates presence cf J? solanacearum irv soil

Figure 9.7 PCR-based assay for the detection of R. solanacearum (Rs) in soil using RS specific primers.

shallow-rooted, short-season crops (Katan and DeVay, 1991; Stapleton, 1994). Solariza-tion does not leave any toxic residue. It is a hydrothermal process dependent for success on moisture of the sample for maximum heat transfer. In more temperate regions soil is covered with clear plastic in order to trap solar radiation and raise the temperature sufficiently to suppress or eliminate soilborne pathogens and pests (Katan 1981; Katan and DeVay, 1991; Kumar et al., 2003). Polyethylene is a suitable cover because it transmits the germicidal component of sunlight. Solarization can be effective against a broad spectrum of soilborne diseases caused by pests such as fungi, nematodes, and bacteria. But the effectiveness of this method is directly linked to climate (Katan and DeVay, 1991). Solarization causes complex biological, physical, and chemical changes that improve plant growth, quality, and yield for up to several years (Stapleton, 1994). Solarization has already proven to be an effective pest control tool for tomato, pepper, and eggplant production in northern parts of Florida and California (Gamliel and Stapleton, 1993). The success of soil solarization is based on the fact that most plant pathogens and pests are mesophiles, which do not produce heat-resistant spores, and they are unable to survive for long periods at high temperature. Death of the organisms at high temperature involves inactivation of enzyme systems, especially respiratory enzymes (DeVay et al., 1990). The greater the temperature, the less time needed to attain a lethal effect. Solarization studies have shown that: solarization reduces or eliminates pathogens and pests prior to planting, crop yields can be significantly increased, and the beneficial effect of solarization can extend through several growing seasons (Afek et al., 1991).

280bp**

Chemical Treatment of Soil and Rhizomes for Bacterial Wilt Control

Hartati and Supriadi (1994) provide evidence of the activity of streptomycin and oxytet-racycline both on the surface and inside tissue of ginger rhizomes soaked in solutions of antibiotics. Similar observations were made by Mulya et al. (1986) for effective bacterial wilt management in ginger. Disease control using commercial chemicals such as antibiotics, fertilizers, and fungicides has not been very successful. Fumigants such as chloropicrin have been used with some success for the control of bacterial wilt of tomato (Enfinger et al., 1979), but in general fumigation is not economically feasible over large areas. Ishii and Aragaki (1963) suggested soil fumigation with methyl bromide at 3 lb/100 ft2 to get good control of bacterial wilt of ginger. The bactericide Kekuling, applied to the rhizosphere of ginger five times during growth of the crop as a wettable powder formulation at dilutions of 1:500 to 1:1200, has been reported to provide good control of bacterial wilt of ginger under field conditions in China (Zhang et al., 1993).

Preplant Rhizome Treatment by Heat

Disinfection of seed pieces prior to planting is an important approach to the control of bacterial wilt of ginger; as noted earlier, contaminated planting material is one of the primary inoculum sources for field infection. Since no chemical and biological control approaches are available for control, a possible alternative is heat treatment to inactivate or kill bacteria, fungi, and nematodes (Janse and Wenneker, 2002). Pathogens are killed either directly by heat or weakened by sublethal heat to the extent that they are unable to damage the crop. Heat can be induced in rhizomes by hot water (Tsang and Shintaku, 1998), hot air, water vapor, solar energy, and microwaves (Kumar et al., 2003). Of these, hot air, hot water, and water vapor have been used for many years as effective treatments for planting material to rid them of various pathogenic microorganisms including nematodes (Colbran and Davis, 1969). Each of the approaches are discussed below.

Heat Induction by Hot Air

Heat inactivation using hot air has had wide application in the control of postharvest disease and insect pests in fruits and vegetables (Couey, 1989). The use of nonsaturated heated air is a potential treatment for disinfection of ginger seed pieces. Exposure of ginger seed pieces to hot air at 75 percent relative humidity (RH) until their center temperature reaches 49°C(112°F) for 30 minutes or 50°C (122°F) for 30 minutes results in minimal injury to the host without an adverse effect either on germination or subsequent growth. Rhizomes harvested from plants grown from seed pieces inoculated with R. solanacearum and subsequently heat treated with hot air at 75 percent RH until their central temperature reached 49°C for 30 minutes were free of the bacterial wilt pathogen. The bacterial wilt pathogen was destroyed in ginger seed pieces treated with hot air at 75 percent RH, and this method is recommended for disinfection (Tsang and Shintaku, 1998). Heat treatment also serves to release the seed piece from dormancy. To be effective, heat treatment must be long enough to penetrate the seed piece to its full depth, but not so prolonged as to be injurious to the host. Selection of similar sized seed pieces is an aid in maintaining uniform thermal gradients in a batch. There are many examples of the use of heat to kill pathogens without affecting the viability of the planting units (Waterworth and Kahn, 1978; Kuniyasu, 1983; Shiomi, 1992; van der Hulst and de Munk, 1992; Dhanvantari and Brown, 1993; Tsang and Shintaku,

1998). In ginger planting of seed pieces after exposure to 50 °C for 30 minutes often resulted in healthy plantlets in Hawaii (Tsang and Shintaku, 1998).

Heat Induction by Hot Water

Soaking of ginger seed in hot water at 50°C for 10 minutes (Nishina et al., 1992; Trujillo, 1963) is the usual preplant preparation in Hawaii. Shorter exposure times give insufficient heat penetration,, and longer soaking periods result in heat injury to the seed piece and growth of stunted crops (Nishina et al. 1992).

Heat Induction by Rhizome Solarization

Disinfection of rhizomes with solar radiation, a method called rhizome solarization, has been developed for bacterial wilt management (Figure 9.8) (Kumar et al., 2003). This is one of the most ecofriendly and energy-efficient methods available for rhizome treatment. Rhizome temperatures of 40 and 50°C were recorded after 1 and 2 hours of solarization from 9:00 a.m. to 11:00 a.m. on a bright sunny day (January to May in India) (Prasheena, 2003). Plants emerging from solarized rhizomes often escape the disease due to in situ killing of the pathogen in the seed rhizome or in the vascular tissue itself (Kumar et al., 2003). Serological evidence for elimination of R. solanacearum from ginger rhizomes has been reported (Prasheena, 2003). When rhizomes are exposed to solar radiation, the rhizome temperature rises especially in the vascular region. Incidentally, the thermal inactivation point for R. solanacearum is 46 to 50°C at 30 minutes of continuous exposure in vitro (Kumar et al., 2003). However, data obtained

Figure 9.8 Effect of rhizome solarization on bacterial wilt incidence in ginger 1. Untreated rhizomes. 2 & 3-Solarized rhizomes.

from studies in vitro with bacterial suspensions are not comparable to the situation where the bacterium is well protected in the vascular tissues of ginger rhizomes. To achieve the requisite temperature inside the vascular tissue or in a site where the pathogen is located, 2 hours of rhizome solarization are sufficient. A temperature of over 49°C was recorded in almost all the locations. The consistency with which the rhizome temperature increases in the rhizome once again confirms the effectiveness of rhizome solarization for heat induction in rhizomes (Prasheena 2003). However, one of the major sources of variability in heat build up vis-à-vis the fate of pathogen, R. solanacearum, is variation in the size and shape of the rhizome. As the size increased the heat build-up was also increased. Larger rhizomes recorded 1 to 3°C higher temperatures than the smaller rhizomes. The variation in the heat build up in the rhizome could be due to the fact that the larger seed rhizome has a larger surface area to trap the sunlight that, in turn, results in a higher temperature in the rhizome (Prasheena, 2003). The relationship between rhizome size and heat build-up has been recorded (Tsang and Shintaku, 1998).

Rhizomes collected from ginger plants emerged from solarized infected rhizomes tested negative for R. solanacearum in NCM-ELISA (Kumar et al., 2003). This result corroborates the finding of Tsang and Shintaku (1998) that the bacterial wilt pathogen was killed due to heat exposure as assayed by PCR using primers specific for R. solanacearum. This could be due to heat killing of the microbial cells on ginger rhizomes including R. solanacearum as the rhizome temperature recorded after 2 hours of rhizome solarization was 50°C. The effect of rhizome solarization on microbial populations has been reported (Prasheena 2003). Tsang and Shintaku (1998) reported that R. solanacearum was eliminated from ginger rhizomes when the rhizome was exposed to heat for 30 minutes at 50°C. Negative results obtained in postenrichment double antibody sandwich (DAS)-ELISA for R. solanacearum in solarized rhizomes confirms that the bacterium does not survive in solarized rhizomes (Table 9.1) (Anila, 2003; Prasheena, 2003). The assay clearly indicates that rhizome solarization is capable of disinfecting the rhizomes infected by R. solanacearum either artificially or naturally. The temperature generated inside the rhizome may have decreased the numbers of viable bacteria in the rhizome. As surface

Table 9.1 Fate of R. solanacearum in solarized ginger rhizomes as detected by DAS-ELISA

A405 value' RS inoculated Uninoculated

Rhizome

A405 value' RS inoculated Uninoculated

Rhizome

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