Biological control of bacterial spot of tomato using hrp mutants of Xanthomonas campestris pv. vesicatoria

Biological Control 41 (2007) 199–206 www.elsevier.com/locate/ybcon

Biological control of bacterial spot of tomato using hrp mutants of Xanthomonas campestris pv. vesicatoria W.P. Moss a, J.M. Byrne a, H.L. Campbell a, P. Ji a, U. Bonas b, J.B. Jones c, M. Wilson b

d,*

a Department of Plant Pathology, Auburn University, Auburn, AL 36849, USA Institut fu¨r Genetik, Martin-Luther-Universita¨t, Halle-Wittenberg, D+06099 Halle, Germany c Department of Plant Pathology, University of Florida, Gainesville, FL 32611, USA d Biology Department, The Colorado College, Colorado Springs, CO 80903, USA

Received 6 July 2006; accepted 18 January 2007 Available online 26 January 2007

Abstract Xanthomonas campestris pv. vesicatoria strain 75-3 hrpG, hrpX, hrpF and hrpE1 mutants were evaluated for control of bacterial spot of tomato under both greenhouse and field conditions. In greenhouse studies, the 75-3 hrp mutants were spray-inoculated onto tomato foliage 48 h prior to inoculation of the wild-type pathogen, X. campestris pv. vesicatoria 75-3, and bacterial spot severity was assessed 10 days after pathogen inoculation. Statistically significant reductions in disease severity relative to the buffer treatment were provided by all four 75-3 hrp mutants. Unexpectedly, however, disease reductions differed significantly among the mutants with the 75-3S hrpG mutant consistently providing the greatest disease suppression (mean reduction in disease severity compared to the buffer treatment of 58%), followed by the hrpX mutant (mean 40%), the hrpF mutant (mean 30%), and the hrpE1 mutant, which provided the least control (mean 21%). Disease control efficacy of these mutants was evaluated under field conditions in Alabama (AL) and Florida (FL). Only the 75-3S hrpG mutant and the 75-3 hrpF mutant provided statistically significant reductions in disease severity compared to the pathogen-only control in all three field trials. In the two AL trials, the 75-3S hrpG mutant was significantly more effective in reducing bacterial spot severity than the previously most effective biological control agents Pseudomonas syringae Cit7 and Pseudomonas putida B56. In the two AL trials, the 75-3S hrpG was as effective as the plant activator acibenzolar-S-methyl in one experiment but less effective in the other experiment. Averaged across all three field trials the 75-3S hrpG mutant provided a mean reduction in foliar disease severity of 76%, compared to the mean of 29% for P. syringae Cit7 averaged across all previous field trials. This study is significant not only for the selection of a biological control agent superior to previously selected strains and comparable in efficacy to the plant activator acibenzolar-S-methyl, but also for the finding that not all hrp mutants of a given bacterial strain are equally effective as biological control agents of that pathogen. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Bacterial spot; Biological control; Tomato; Mutant; hrpG; hrpE1; hrpF; hrpX; Xanthomonas campestris pv. vesicatoria; Lycopersicon esculentum

1. Introduction Bacterial spot of tomato (Lycopersicon esculentum), a serious disease affecting both foliage and fruit (Jones, 1991), is caused by Xanthomonas campestris pv. vesicatoria (syn. Xanthomonas euvesicatoria Jones et al., 2004). The disease is particularly important in the southeastern United

*

Corresponding author. Fax: +1 719 389 6940. E-mail address: [email protected] (M. Wilson).

1049-9644/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2007.01.008

States (Davis et al., 1998). Although bacterial spot may not reduce the total number of fruit, economic losses result from unmarketable infected fruit, sun scalded fruit due to defoliation, and reduced numbers of fruit in the USDA large size category which brings the highest return to growers (Pohronezny and Volin, 1983). Traditional control measures for bacterial spot include the application of bactericides, such as copper hydroxide, in combination with ethylene bis-dithiocarbamate (EBDC). Inadequate efficacy of the conventional chemical agents, particularly when copper-resistant pathogen strains are prevalent, make

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alternative or complementary approaches for management of bacterial spot highly desirable. In recent years there has been increasing interest in the biological control of bacterial plant diseases using either bacteriophages (Flaherty et al., 2000; Flaherty et al., 2001; Balogh et al., 2003; Obradovic et al., 2004) or naturally-occurring saprophytic bacteria (Backman et al., 1997; Lindow and Wilson, 1999; Wilson, 2004; Wilson and Backman, 1999). However, while bacterial biological control agents have been developed commercially for crown gall, caused by Agrobacterium tumefaciens; fire blight, caused by Erwinia amylovora; and frost injury, caused by ice-nucleation active (Ice+) strains of Pseudomonas syringae (Wilson, 2004), to date, no effective bacterial biological control agent of bacterial spot of tomato is available. In general, studies on biological control of diseases caused by Xanthomonas spp. using naturally-occurring saprophytic bacteria are limited compared to those on biological control of P. syringae pathovars and E. amylovora. Examples of studies on the biological control of Xanthomonas pathogens include: X. axonopodis pv. allii (Gent and Schwartz, 2005); X. campestris pv. campestris (Assis et al., 1996; Massomo et al., 2004; Wulff et al., 2002); X. campestris pv. dieffenbachiae (Fukui et al., 1999a); X. campestris pv. glycines (Rukayadi et al., 2000); X. campestris pv. graminis (Schmidt, 1988a and b); X. campestris pv. malvacearum (Verma and Singh, 1989); X. campestris pv. phaseoli (Romeiro et al., 2005); X. campestris pv. translucens (Stromberg et al., 2000); X. campestris pv. vignaeradiatae (Bora et al., 1993); Xanthomonas albilineans (Zhang and Birch, 1997); and X. oryzae pv. oryzae (Hsieh and Buddenhagen, 1974; Arya and Parashar, 2002). Biological control of X. campestris pv. vesicatoria has been reported under both greenhouse (Dianese et al., 2003; El-Hendawy et al., 2005) and field conditions (Byrne et al., 2005; Ji et al., 2006). In the field, low to moderate, but statistically significant, suppression of bacterial spot of tomato was reported in several trials using foliar applications of the naturally-occurring saprophytic bacteria Pseudomonas syringae Cit7 and Pseudomonas putida B56 (Byrne et al., 2005) or rhizosphere applications of non-pathogenic plant growth-promoting bacteria (PGPR) (Ji et al., 2006). However, despite novel approaches, such as combinations of foliar biological control agents and PGPR (Ji et al., 2006) or mixtures of biological control agents (Fukui et al., 1999b), the efficacy of naturally-occurring saprophytic bacteria for biological control of Xanthomonas diseases generally has been disappointing. An alternative to the use of naturally-occurring saprophytic species of bacteria is the use of naturally-occurring avirulent or nonpathogenic strains of either the cognate pathovar or pathovars of a different host. There has been a long history of experimentation with naturally-occurring avirulent mutants of Ralstonia solanacearum (Chen et al., 1981; Trigalet and Trigalet-Demery, 1990) and Erwinia amylovora (Goodman, 1967; McIntyre et al., 1973), but

relatively few experiments with X. campestris. Naturallyoccurring avirulent pathovars of X. campestris have been evaluated for control of a few X. campestris pathogens: for example, X. campestris pv. malvacearum was evaluated as a possible biological control agent of black rot of cabbage, caused by X. campestris pv. campestris (Jetiyanon, 1994); and X. campestris pv. raphani has been used in an attempt to control X. campestris pv. vesicatoria in tomato (Sahin and Miller, 1997). However, in general, these avirulent pathovars and naturally-occurring non-pathogenic strains have been no more effective than the saprophytes. Near-isogenic, nonpathogenic mutants of a bacterial plant pathogen for the purpose of biological control can be generated through the deletion of genes involved in pathogenicity/virulence (Lindemann, 1985; Napoli and Staskawicz, 1985); specifically through the mutation of one or more hypersensitive response and pathogenicity (hrp) genes (Wilson et al., 1998). Such hrp mutants have been evaluated as potential biological control agents of the pathogenic parent in several pathosystems: a hrpO mutant of R. solanacearum significantly reduced bacterial wilt severity under greenhouse conditions (Frey et al., 1994); hrp mutants of E. amylovora protected apple shoots against fire blight following wound inoculation in the greenhouse (Tharaud et al., 1993, 1997; Faize, Brisset et al., 1999; Faize, Tharaud et al., 1999); and a hrpS mutant of P. syringae pv. tomato provided statistically significant control of bacterial speck under field conditions (Wilson et al., 2002). Substantial progress has been made in understanding hrp gene expression and regulation in X. campestris pv. vesicatoria (Bu¨ttner and Bonas, 2002). The two regulatory genes hrpG (Wengelnik et al., 1996) and hrpX (Wengelnik and Bonas, 1996) regulate expression of the hrp structural genes, such as hrpF (Bu¨ttner et al., 2002; Huguet and Bonas, 1997; Rossier et al., 2000) and hrpE1 (Weber and Koebnik, 2005). The products of the hrp structural genes are involved in type III secretion system (TTSS) structure and function or are effector proteins, secreted into the plant cell (Bu¨ttner and Bonas, 2002). In this study, X. campestris pv. vesicatoria strain 75-3 with mutations in either regulatory genes, hrpG or hrpX, or in structural genes, hrpE1 or hrpF, were evaluated for ability to reduce bacterial spot severity when applied in advance of the wild-type parental pathogen strain under both greenhouse and field conditions. Preliminary results of this study have been presented elsewhere (Moss 2000; Moss et al., 1997, 1998a, 1998b). 2. Materials and methods 2.1. Bacterial cultures Xanthomonas campestris pv. vesicatoria strain AD17 was isolated from symptomatic tomato foliage collected in Alabama (Dianese, 1997) (Table 1). X. campestris pv. vesicatoria strain 75-3 was isolated from tomato in Florida (Minsavage et al., 1990) (Table 1). In this study, a

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Table 1 Bacterial strains and plasmids used in this study Strain or plasmid

Characteristicsa

Reference or source

E. coli DH5a HB101

F recA u80dlacz DM15 F recA; SmR

BRL, Bethesda

X. campestris pv. vesicatoria AD17 Race T1, CuS 75-3 Race T1, RfR, CuS 75-3S Spontaneous SmR mutant of 75-3 75-3 hrpE1 75-3 hrpE::Tn5 #84; RfR, KmR 75-3 hrpF 75-3 hrpF::Tn5 #13; RfR, KmR 75-3S hrpG 75-3S hrpG::Tn3-gusA; RfR, KmR 75-3 hrpX 75-3 hrpX::Xsp; RfR, SpR (75-3 hrpX is called 75X in Wengelnik and Bonas, 1996) 75-3 Tn5#16 75-3::Tn5-16; 75-3 Tn5 opgHXcv mutant

Dianese, 1997 Minsavage et al., 1990 This study Bonas et al., 1991 Bonas et al., 1991 This study Wengelnik and Bonas, Minsavage et al., 2004

Pseudomonas spp. B56 Cit7

P. putida isolated from tomato foliage P. syringae isolated from orange

Byrne et al., 2005 Lindow, 1985

Plasmids pXV1::Tn1006 pRK2013

hrpG::Tn3-gusA derivative of pXV1; TcR, KmR ColE1 replicon; TraRK+; Mob+; KmR

Wengelnik et al., 1996 Figurski and Helinksi, 1979

a

RfR, KmR, SmR, SpR, and TcR indicate resistance to rifampicin, kanamycin, streptomycin, spectinomycin, and tetracycline, respectively.

spontaneous streptomycin resistant mutant of strain 75-3, designated 75-3S, was selected and the pathogenicity of this strain was compared to that of 75-3. Nonpathogenic 75-3 hrpX, hrpF, and hrpE1 mutants (Bonas et al., 1991) (Table 1) were supplied by Bonas. Mutant 75-3::Tn5#16, which is severely attenuated in pathogenicity (Minsavage et al., 2004), was supplied by Minsavage. All cultures were stored at 80 °C in tryptic soy broth (TSB) amended with 20% (v/v) glycerol. A nonpathogenic hrpG mutant of 75-3S was created by marker exchange mutagenesis using pXV1::Tn1006 which carries a Tn3-gusA insertion in hrpG (Wengelnik et al., 1996) (Table 1). Triparental mating and selection of the correct mutant were carried out as described previously (Wengelnik et al., 1996; Schulte and Bonas, 1992). The ability of putative 75-3S hrpG mutants to induce a hypersensitive response (HR) was tested according to standard methodology. A hrpG mutant of 75-3S was selected and stored at 80 °C in TSB amended with glycerol (20%, v/v). 2.2. Greenhouse experiments Tomato seedlings (cv. Agriset 761) were grown as described previously (Byrne et al., 2005; Dianese et al., 2003; Ji et al., 2006). Six to seven-week-old tomato plants were incubated in the greenhouse at high relative humidity (RH) for 24 h prior to application of 75-3R hrp mutants. Bacteria were grown on TSA amended with Rf (100 lg/ ml) for 24 h at 28 °C, suspended in potassium phosphate buffer (0.01 M; pH 7.0), and cell density was adjusted turbidimetrically to the appropriate concentration. The 75-3 hrp mutants at a concentration of 107 CFU/ml were spray-inoculated onto the abaxial and adaxial leaf surfaces of four replicate tomato plants until runoff using hand-trig-

ger sprayers. The pathogen X. campestris pv. vesicatoria 753 at a concentration of 106 CFU/ml was inoculated 48 h after application of the 75-3 hrp mutants. The inoculated plants were incubated in the greenhouse under disease-conducive conditions until symptom appearance (Byrne et al., 2005; Dianese et al., 2003; Ji et al., 2006). The experiment was conducted four times and all experiments used a randomized complete block design with a minimum of six blocks. A buffer-only and pathogen-only treatment were included in each experiment. Ten tomato leaflets per replicate plant were sampled randomly ten days after inoculation with the pathogen. Disease data were collected analyzed as described in previous studies (Byrne et al., 2005; Dianese et al., 2003; Ji et al., 2006). In brief, lesions on individual leaflets were counted and leaflet area determined; disease severity was quantified as lesions/leaflet area; disease severity data were log transformed; and log-transformed severity data were subjected to analysis of variance using the ANOVA or GLM procedures of the Statistical Analysis System (SAS Institute, Gary, NC) and means comparisons using Duncan’s multiple range test. Reduction in disease severity compared to the buffer-only control was calculated according to the formula: Disease reductionð%Þ ½Disease severitycontrol  Disease severitytreatment  ¼  100 Disease severitycontrol Calculation of a percentage disease reduction for the biological treatments allows a mean disease reduction to be calculated across replicate experiments with different levels of disease and allows the efficacy of the biological

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control agents to be compared to those examined in previous studies (Byrne et al., 2005; Dianese et al., 2003; Ji et al., 2006).

3. Results

2.3. Field experiments

The 75-3 hrpX, hrpE1 and hrpF mutants and the 753S hrpG mutant were tested in a greenhouse for their ability to reduce the severity of bacterial spot of tomato caused by the parental pathogen strain 75-3. In addition to the conventional ‘‘pathogen-only’’ control, a buffer treatment (i.e., buffer followed by pathogen 48 h later) was also included (Table 2) since occasionally small reductions in disease severity have been attributable to phosphate buffers. The 75-3S hrpG mutant and the 753 hrpX mutant provided significant reductions in disease severity compared to the buffer treatment in all four experiments; the 75-3 hrpF mutant provided significant reductions compared to the buffer-treatment in three out of four experiments; and the 75-3 hrpE1 mutant provided significant reductions compared to the buffertreatment in two out of four experiments (Table 2). Of the hrp mutants, the 75-3S hrpG mutant provided the greatest mean disease reduction (58% compared to the buffer treatment [or 63% compared to the pathogen-only control]), followed by the 75-3 hrpX mutant (mean  40%), the 75-3 hrpF mutant (mean  30%), and the 75-3 hrpE1 mutant provided the least control (mean  21%) (Table 2). Xanthomonas campestris pv. vesicatoria 75-3 Tn5#16, a mutant of significantly reduced virulence and aggressiveness, reduced disease severity significantly in all three experiments in which it was included. In one of the three experiments the disease severity in the 75-3 Tn5#16 treatment was significantly less than the disease severity in the 75-3S hrpG experiment, while in the other two experiments the disease severity in the 75-3 Tn5#16 and 75-3S hrpG treatments were not significantly different. Despite these interesting results, mutant Tn5#16 was not incorporated in the field trials because it is not completely non-pathogenic, being only attenuated in virulence.

Field experiments were conducted in Shorter, Alabama, during the fall of 1997 and during the summer of 1998 and in Gainesville, Florida. Tomato field plots were established and maintained as described previously (Byrne et al., 2005; Ji et al., 2006; Wilson et al., 2002). A randomized complete block design with 5 blocks was used for both field experiments, where each block consisted of a ten-plant row. The 75-3 hrpG, hrpX, hrpF, and hrpE1 mutants were grown on TSA amended with Rf (100 lg/ml) for 24 h at 28 °C prior to inoculation. Bacteria were suspended in sterile potassium phosphate buffer (0.1 M; pH 7.4), adjusted to a concentration of 108 CFU/ml and applied to tomato plants at a rate of approximately 4 L/50 plants using a CO2-powered backpack sprayer. Two other bacterial strains, P. putida B56 and P. syringae Cit7, which provided moderate control of bacterial spot under field conditions (Byrne et al., 2005), were included for comparison with 75-3 hrp mutant treatments. Chemical treatments, copper/maneb (Kocide DF and Manex; Griffin Corp., Valdosta, GA) and acibenzolar-S-methyl (Actigard; CGA245704-50WG; Novartis Corp., Greensboro, NC) (Louws et al., 2001), were mixed and applied according to label specifications. Chemical and biological treatments were applied weekly for a period of ten weeks. In each experiment, a set of control plants was left untreated. Field plots were inoculated once, approximately 6 weeks after transplant, with a suspension of X. campestris pv. vesicatoria strain AD17 at a concentration of 107 CFU/ml to ensure the development of adequate disease. Disease was assessed once approximately 2 weeks after pathogen inoculation. Foliar disease severity was determined by counting lesions on each of 20 randomly sampled leaflets per replicate row. Disease severity data were analyzed as described above.

3.1. Greenhouse experiments

Table 2 Suppression of bacterial spot on tomato by X. campestris pv. vesicatoria 75-3 hrp mutants and 75-3 Tn5#16 mutant under greenhouse conditions Treatmenta

Experiment #1 b

Pathogen-only control Phosphate buffer 75-3S hrpG 75-3 hrpX 75-3 hrpF 75-3 hrpE1 75-3R Tn5#16 a b c d

Dis sev

Reduction

0.40c a 0.25 b 0.15 c 0.18 c 0.27 b 0.24 b —

— — 40% 28% 0 4% —

d

Experiment #2

Experiment #3

Experiment #4

Dis sev

Reduction

Dis sev

Reduction

Dis sev

Reduction

8.08 6.88 2.49 3.51 4.11 5.05 2.48

— — 64% 49% 40% 27% 69%

1.61 1.86 0.54 0.85 1.15 1.18 0.59

— — 71% 54% 38% 37% 63%

1.85 1.79 0.79 1.26 1.02 1.52 0.71

— — 56% 30% 43% 15% 62%

a a d c bc b d

a a c b b b c

a a de bc cd ab e

Mean reduction

— — 58% 40% 30% 21% 65%

Treatments were applied to tomato plants (cv. Agriset 761) 48 h prior to pathogen inoculation. Disease assessed 10 days after inoculation with the pathogen. Disease severity was quantified as lesions/cm2 leaf area. Means in a column followed by the same letter are not significantly different according to Duncan’s multiple range test at P = 0.05. Disease reduction (%) compared to buffer treatment.

W.P. Moss et al. / Biological Control 41 (2007) 199–206

3.2. Field experiments Disease control efficacy of the 75-3 hrp mutants was evaluated under field conditions in Alabama (AL) in 1997 and 1998 in comparison with the biological control agents P. syringae Cit7 and P. putida B56, which previously provided low to moderate disease control in repeated field trials (Byrne et al., 2005); a copper/EBDC formulation; and acibenzolar-S-methyl (Actigard), a plant activator (Louws et al., 2001). Disease control efficacy of the 75-3 hrp mutants was also evaluated in Florida (FL), but the other treatments were not included in that trial. In the 1997 trial in AL, three biological agents, 75-3S hrpG, 75-3 hrpF, and P. syringae Cit7 reduced disease severity significantly compared to the buffer treatment (Table 3). In this experiment the disease severity in the 75-3S hrpG mutant treatment was significantly less than the disease severity in the plants treated with the other hrp mutants or the other two biological control agents Cit7 and B56; was not significantly different from the copper/EBDC treatment; but was significantly greater than the disease severity in the acibenzolar-S-methyl treatment (Table 3). In the 1998 trial in AL, all six biological agents reduced disease severity significantly compared to the pathogen-only control (Table 3). The disease severity in the 75-3S hrpG mutant treatment was significantly less than the disease severity in all other treatments, except for the acibenzolar-S-methyl treatment with which there was no significant difference (Table 3). In the trial in FL, the disease severities in the 75-3S hrpG, hrpE1, and hrpF treatments were significantly less than the disease severity in the pathogen-only control, but not significantly different from each other (Table 3). Across the three field trials, the 75-3S hrpG mutant provided a mean reduction in foliar disease severity of

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76%, compared to the most effective treatment, acibenzolar-S-methyl, which provided a mean reduction of 80% (Table 3). 4. Discussion The X. campestris pv. vesicatoria 75-3S hrpG mutant provided significant control of bacterial spot of tomato under both greenhouse and field conditions, with mean reductions in disease severity of 58% and 76%, respectively. The level of control provided by this mutant was significantly greater than that provided by any of the other three hrp mutants (75-3 hrpX, hrpF, or hrpE1 mutants) mutants; the two biological control agents, P. syringae Cit7 or P. putida B56; or the copper/EBDC combination. In one field experiment, the level of control achieved by the 75-3S hrpG mutant was not significantly different from the plant activator acibenzolar-S-methyl (Actigard), the most effective chemical agent currently available (Louws et al., 2001). The previous most effective bacterial biological control agent of bacterial spot, P. syringae Cit7, provided a mean of 29% reduction in disease severity when averaged across nine different field experiments in different locations across North America (Byrne et al., 2005). While statistically significant and reasonably consistent, such a level of control is nevertheless inadequate to justify the incorporation of P. syringae Cit7 into current bacterial spot control practices in commercial production. In contrast, the level of control provided by X. campestris pv. vesicatoria strain 75-3S hrpG mutant shows great promise and deserves to be tested more extensively. Despite the fact that the mean level of disease reduction by 75-3S hrpG would undoubtedly be less when averaged over additional field experiments, due to the innate variability of biological control, this is possibly one of the most exciting developments in biological control of foliar bacterial diseases of

Table 3 Suppression of bacterial spot on tomato by X. campestris pv. vesicatoria 75-3 hrp mutants under field conditions Treatmenta

Experiment #1 (fall 1997—AL) b

Pathogen-only control Phosphate buffer 75-3S hrpG 75-3 hrpX 75-3 hrpE1 75-3 hrpF B56 Cit7 Kocide/Manex Acibenzolar-S-methyl

Dis sev

Reduction

21.26d a 21.00 a 6.46 c 17.26 a 15.36 ab 11.33 b 14.67 ab 10.93 b 7.26 c 4.33 d

— 1.2% 69.6% 18.8% 27.8% 46.7% 31.0% 48.6% 65.9% 79.6%

e

Experiment #2 (summer 1998—AL) b

Experiment #3 (FL) c

Dis sev

Reduction

Dis sev

Reduction

111.87 a NT 23.89 e 47.86 cd 73.49 b 42.27 d 70.32 b 59.63 bc 43.44 cd 20.87 e

— NT 78.6% 57.2% 34.3% 62.2% 37.1% 46.7% 61.2% 81.3%

0.394 NT 0.078 0.216 0.174 0.098 NT NT NT NT

— NT 80.2 45.2 55.8 75.1 NT NT NT NT

a b ab b b

Mean reduction f

— — 76.1 40.4 39.3 61.3 34.0 47.6 63.6 80.4

NT, not tested. a Treatments were applied to tomato plants weekly. b Disease severity was assessed as lesions/leaflet. c Disease severity was assessed as lesions/cm2. d Means in a column followed by the same letter are not significantly different according to Duncan’s multiple range test at P = 0.05. e Disease reduction (%) compared to buffer treament. f Disease reduction (%) compared to the pathogen-only treatment.

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tomato in several years of research (Byrne et al., 2005; Dianese et al., 2003; Ji et al., 2006; Wilson et al., 2002). This optimism, however, should be tempered by the possibility that a genetically modified organism (GMO) might face issues of acceptability with the farming community or public. The observation that the hrp mutants provided significantly different levels of disease reduction was somewhat unexpected, though not without precedent: of the three hrp mutants of P. syringae pv. tomato strain DC3000 tested, only the hrpS mutant provided significant control of bacterial speck under field conditions (Wilson et al., 2002); and of the two hrp mutants of E. amylovora tested, one mutant was significantly more protective than the other (Faize, Brisset et al., 1999; Faize, Tharaud et al., 1999). Perhaps the most interesting observation across these different studies is that hrp regulatory mutants were more effective than the TTSS structural mutants: hrpG in X. campestris pv. vesicatoria (this study); hrpS in Pseudomonas syringae pv. tomato (Wilson et al., 2002); and PMV6046 regulatory mutant in Erwinia amylovora (Faize, Brisset et al., 1999). The significance of this observation is not entirely understood but undoubtedly relates to the mechanism of control by these hrp mutants and is the subject of current study. In the case of X. campestris pv. vesicatoria it had been assumed that the hrp mutants would function through pre-emptive competitive exclusion as described with near-isogenic Ice and Ice+ P. syringae strains (Lindow, 1985; Wilson and Lindow, 1994) and, therefore, that any differences in biological control would relate to minor differences in ability to colonize the plant surface, however, this does not appear to be the case (Moss, 2000). We currently hypothesize that the hrp mutants induce a host defense response and, in the case of X. campestris pv. vesicatoria, the magnitude of the defense response mounted by the plant is greatest in response to the regulatory 75-3S hrpG mutant and least in response to the structural 75-3 hrpE1 mutant (Wilson et al., 2006). The studies currently underway to elucidate the nature of these defense responses should provide valuable information about the importance of the hrpG regulon in pathogenicity of X. campestris pv. vesicatoria and about the nature of the host defense responses in a compatible/ susceptible interaction that does not involve an R-avr interaction (Wilson et al., 2006). The induction of an effective host defense response by the hrpG mutant would also have important implications for control of bacterial diseases. The broad pathosystem efficacy of the plant activators acibenzolar-S-methyl (Actigard) and harpin (Messenger) suggests that 75-3S hrpG may also protect against other foliar diseases of tomato, such as bacterial speck, but this has not yet been tested. There also exists the possibility that 75-3 hrpG mutant, or a hrpG mutant of the cognate pathogen, could provide superior protection against other Xanthomonas diseases, such as bacterial blight of rice, caused by X. oryzae pv. oryzae.

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