May, 1987
There are over 125 pathovars of X. campestris described, and the only means to differentiate between them is by pathogenicity testing [6]. Some strains of X. campestris have extended host ranges to more than one host, usually to other members of the same plant family, with few exceptions [157]. The host range can sometimes overlap onto another host for which a different X. campestris pathovar has been described. For instance, X. c. pv. alfalfae is a pathogen of the legumes alfalfa, bean, and pea, but strains exist of X. c. pv. phaseoli and X. c. pv. pisi which have host ranges limited to bean and pea, respectively. The relatedness between these strains at the subspecific level is unknown, except for their host ranges. In another instance, strains of X. c. pv. malvacearum have been isolated from the malvaceous hosts, cotton and hibiscus [110,410]. Although given the same pathovar designation because they were each isolated from plants of the Malvaceae family, their relatedness is unknown. Similar situations, where the relationship between strains is unclear, exists among other pathovars of X. campestris [6]. The purpose of this investigation was to verify the pathogenicity and host range of all stock strains used in this dissertation, to examine the relationships between apparently related strains of X. campestris in terms of host range specificity, and to attempt to find a biochemical test diagnostic for one or more pathovars used in these studies.
Bacterial Strains and Host Plants
Bacteria used in this investigation consisted of pathovars of X. campestris which were pathogenic to either the Leguminosae or Malvaceae plant families. The pathogens of leguminous host plants consisted of X. campestris pvs. alfalfae, cyamopsidis, glycines, phaseoli, pisi, and vignicola isolated from alfalfa, guar, soybean, kidney bean, pea, and cowpea, respectively. The pathogens of malvaceous host plants consisted of strains of X. campestris pv. malvacearum isolated from cotton or hibiscus. The strains of X. campestris used in this study are shown in Table 3-1. The legume host plants used in this investigation included Medicago sativa cv. FL-77, Glycines max cv. Evans, Phaseolus vulgaris cv. California Light Red, and Vigna unguiculata cv. California Blackeye #5. The malvaceous hosts included three cultivars of Hibiscus rosa-sinensis and eight cultivars of Gossypium hirsutum. One of the cultivars of H. rosa-sinensis was cv. Brilliant Red; the other two were not named, but chosen to represent diversity in the species. The cotton cultivars consisted eight cultivars developed for differentiating races of X. c. pv. malvacearum, of which two were cv. 101-102B, and cv. Gregg [109]. The other cultivars were cv. Acala 44 (no resistance to cotton strains), and five cultivars derived from cv. Acala 44 crossed to obtain single gene resistance to the cotton strains of X. c. pv. malvacearum, the cultivars being Acala B1, B2, B3, B5, and BIN [110].
Plant Inoculations
Pathogenicity tests were conducted by pressure infiltrating bacterial suspensions into leaf tissue with a blunt-end syringe, and incubating the plants in 28°C or 30°C growth chambers until symptom were expressed. The leguminous host plants were incubated at 28°C and symptoms were recorded after one week. The malvaceous hosts were incubated at 30°C and symptoms were recorded after a two week period. Pressure infiltration facilitated rapid screening of plant reactions, but was not representative of natural inoculation. Because alfalfa leaves were small in size, infection was by spray inoculation. Bacterial suspensions were prepared by centrifuging overnight cultures of X. campestris, resuspending them in 0.7% NaCl to an approximate optical density (OD600nm) of 0.3 for plant inoculation. These pathogenicity tests were repeated at least once.
Biochemical Differentiation
As X. campestris is defined [6], the biochemical characteristics for gelatin and starch hydrolysis may be variable for this species. Other biochemical characteristics which are consistent with this species includes xanthomonadin production, mucoid growth, and esculin and casein hydrolysis. These above mentioned characteristics were determined using standard methods [423,439]. Additionally, biochemical tests for production of cellulase [418], lecithinase, lipase, and pectinases were included. Hildebrand's medium prepared at three pHs (4.5, 7.0, and 8.5) was used to detect pectolytic enzyme activity. In each of these three media, sodium polypectate was used as the carbon source. The pH 5 medium included only pectin as the sole carbon source. These tests were each repeated once.
Polyacrylamide Gel Electrophoresis
Bacteria were grown overnight at 30°C in a peptone-glycerol broth. Cells were adjusted to a 0.3 optical density (OD600nm), and 1.5 mls of cells were centrifuged, washed once in water, and resuspended in 50 µl 10% sorbitol. 50 µl solubilization buffer (90 mM Tris (pH 6.8), 20% glycerol, 4% SDS, 10% ß-mercaptoethanol, and .002% bromophenol blue) was added, the suspension was boiled for 5 min. Approximately 10 mg protein (10 ul solution) was added to a 10% resolving acrylamide gel [438], and samples were electrophoresed in a 20 cm x 17 cm x 1.5 mm vertical gel unit at 10 V/cm and 15°C until the dye reached the bottom of the gel. Biorad low-range SDS-PAGE standards were used as molecular weight markers. Gels were stained with 0.1% Coomassie blue in 42% methanol and 17% glacial acetic acid. The gel was destained in 30% methanol and 10% glacial acetic acid. Electrophoresis was only performed once. A photograph of the gel was taken using Polaroid type 55 film.
Pathogenicity Tests
All of the strains of X. c. pv. phaseoli, with one exception, gave the expected phenotypes on the legume hosts tested. The exception, strain B5B, was found to be non-pathogenic all four hosts tested. The remaining six strains were pathogenic on kidney bean (Table 3-2), giving watersoaked lesions on inoculated leaves after four days. The lesions extended from the inoculation sites giving the appearance that the bacterium was spreading throughout the leaf.
The strains of X. c. pv. phaseoli which were found pathogenic on kidney bean were not found to be pathogenic on cowpea, soybean, or alfalfa. In cowpea and soybean, a hypersensitive reaction was apparently elicited, resulting in dry necrotic lesions at the inoculation sites. In soybean the lesion was slightly chlorotic, whereas for cowpea, a wine red lesion occurred. However, X. c. pv. phaseoli strain XP2 did not elicit the wine red color on cowpea (Table 3-2). No response was observed on alfalfa from the spray inoculations with X. c. pv. phaseoli.
The strains of X. c. pv. vignicola were pathogenic on cowpea, resulting in irregularly shaped lesions which had a tearing, or 'shothole' appearance at the inoculation site. On soybean, the incompatible response appeared dry and collapsed. Again, no response was observed from spray inoculation on alfalfa (Table 3-2).
On kidney bean, a range of different responses occurred from inoculations with X. c. pv. vignicola. For two of the strains, A81-331 and C1, an incompatible response appeared as a dry collapsed lesion. For the other strains of X. c. pv. vignicola, there appeared to be some slight water-soaking in the tissue about the periphery of the inoculation site. In two of these instances, for strains 8238 and SN2, the 'shothole' effect as seen on cowpea was observed.
The strains of X. c. pv. glycines appeared to be pathogenic on both soybean and kidney bean, but not on cowpea or alfalfa (Table 3-2). The pathogenic responses appeared as watersoaked lesions, with the lesion being slightly chlorotic in soybean. The incompatible response in cowpea appeared as wine red in color like that seen with X. c. pv. phaseoli.
From spray inoculations on alfalfa, only X. c. pv. alfalfae, resulted in a pathogenic response. After 7 days, small water-soaked leaf spots which later turned necrotic appeared. X. c. pv. alfalfae was also pathogenic on kidney bean, and appeared so slightly on soybean. There were small water-soaked spots which occurred at the inoculation sites on soybean. The wine red incompatible response was observed on cowpea.
A strain of X. c. pv. malvacearum, which was not a pathogen of legumes, resulted in only a slight tissue discoloration at the inoculation site on bean and soybean. A null reaction, as observed with the water-only control, was observed on cowpea. The water-only control also resulted in a null reaction on the other hosts tested. A summary of the phenotypes observed for the inoculation experiments is given in Table 3-3.
In separate inoculation tests, a strain of X. c. pv. cyamopsidis was not found pathogenic on alfalfa, kidney bean, cowpea, or soybean and a X. c. pv. pisi strain was not found pathogenic on kidney bean. Overall, most of the reactions observed conformed to those reported in the literature, with some exceptions. Variation was evident among strains of a given pathovar in addition to that between pathovars. The X. c. pv. alfalfae strains appeared to have overlapping host ranges, which extended to bean and pea in addition to alfalfa. The kidney bean cultivar, California Light Red, appeared susceptible to a representative strain from each of the X. campestris pathovars of legumes tested, with the exception of X. c. pv. cyamopsidis.
Various phenotypic responses were observed on the cotton and hibiscus cultivars with the X. c. pv. malvacearum strains tested. In general, it appeared that those strains of X. c. pv. malvacearum derived as pathogens from cotton were pathogenic to one of the hibiscus cultivars tested. None of the X. c. pv. malvacearum strains derived as pathogens from hibiscus were pathogenic on the cotton cultivars in these tests.
From the inoculation tests on the cotton host differentials with the cotton derived strains of X. c. pv. malvacearum, it appeared that three races of the pathogen were being used. All of these strains were pathogenic on Acala 44 (susceptible host), but could be differentiated into races by the other cotton lines containing different resistance gene backgrounds. The X. c. pv. malvacearum strain N was pathogenic on all the cotton lines tested, while strain H was only pathogenic on Acala 44. Strains FL79 and TX84 appeared to be the same race because they were pathogenic on the same six out of eight cotton lines tested (Table 3-4). However, these two strains differed in reactions on the three hibiscus cultivars inoculated. Strain FL79 appeared pathogenic on the three cultivars, but TX84 gave a null response similar to the water-only control. The two other X. c. pv. malvacearum strains (N and H) appeared identical in reaction with the three hibiscus cultivars, giving pathogenic responses to 2 cultivars, and a null response on the remaining cultivar (Table 3-4).
With the eight X. c. pv. malvacearum strains derived from hibiscus, two gave null reactions on all of the cotton and hibiscus cultivars tested (Table 3-4). The other strains derived from hibiscus were pathogenic on at least two of the three hibiscus cultivars tested, but were not pathogenic on any of the cotton cultivars. In a separate experiment, one of the strains (M84-11) did appear to give pathogenic symptoms on Acala 44, while another strain (83-4244) gave a hypersensitive response. In this instance the mean temperature was at 25 C, rather than 30 C.
An additional strain tested, X. c. pv. esculenti strain 84-1093, which was a pathogen derived from okra, gave a null reaction on seven of eight cotton cultivars and one of three hibiscus cultivars. The other cotton or hibiscus cultivars gave hypersensitive responses (Table 3-4).
Biochemical Differentiation
All of the strains tested were mucoid on NYGA medium, produced xanthomonadin, and hydrolyzed esculin and casein. All of the strains tested also appeared to be positive for starch and gelatin hydrolysis, and production of lipase (Table 3-5). Six of the seven X. c. pv. vignicola strains, and one strains of X. c. pv. phaseoli appeared to hydrolyze gelatin slower than the other X. campestris strains. Cellulase activity, indicated by pitting of the solid medium, was observed for all seven of the pathovars tested. However, not all strains of X. c. pv. phaseoli appeared to have cellulase activity, and X. c. pv. alfalfae strains were weaker in this activity. None of the strains tested exhibited pectolytic activity at pH 4.5, suggesting a lack of polygalacturonase activity. At pH 7.0 and 8.5, pectolytic activity was observed for all strains of X. c. pv. cyamopsidis, X. c. pv. glycines, X. c. pv. pisi, and X. c. pv. vignicola. Only one of the 13 X. c. pv. phaseoli strains tested (SC-3B) had pectolytic activity. None of the strains degraded pectin as a carbon source, only sodium polypectate.
Polyacrylamide Gel Electrophoresis
Total protein was extracted from 18 strains, representing seven different pathovars of X. campestris. The pathovars included were X. c. pv. alfalfae, X. c. pv. cyamopsidis, X. c. pv. glycines, X. c. pv. malvacearum, X. c. pv. phaseoli, X. c. pv. pisi, and X. c. pv. vignicola. All of the pathovars, except X. c. pv. malvacearum (Fig. 3-1, lanes P and Q) are known pathogens of legumes. One of the X. c. pv. phaseoli strains consisted of a biochemical variant of this pathovar, X. c. pv. phaseoli var. fuscans (Fig. 3-1, lane J). One of the X. c. pv. malvacearum strains were derived from hibiscus, rather than from cotton (not shown).
In general, there were few observable differences in the protein banding patterns for the X. campestris strains. Some minor differences in banding patterns between different pathovars were observed, but variation to the same extent was also present within a given pathovar.
The phenotypes observed from the X. campestris host range study conformed to expectations, in most cases. However, it was evident that variation in strains associated with a given pathovar could occur, as shown by the race specificity variation In the legume study, it was interesting to note that different hosts gave different phenotypic responses to different incompatible pathovars of X. campestris. These different incompatible phenotypes suggest that different host resistance genes may be involved in each of these incompatible interactions. Although the hypersensitive reaction is commonly considered to be the general resistance mechanism of plants against bacteria, these data suggest a more specific response, unique to each incompatible reaction [106,148]. No formal studies on the hypersensitive reaction were implemented, so the significance of this observation will require further investigation. Differences in the phenotypes which may account for this are time of recognition, cell number, electrolyte leakage, or other bacteria- or plant-conditioned responses. Likewise, a hypersensitive reaction may have occurred for instances where a null response was observed [446].
The results from inoculation tests on the malvaceous hosts suggested that those strains of X. c. pv. malvacearum derived from cotton are potential pathogens of hibiscus, whereas those derived from hibiscus are generally restricted to hibiscus. However, in one experiment, it was found that one of the hibiscus strains caused watersoaked lesions on Acala 44 when the night temperatures were lowered. This observation indicates that some of the interactions may be temperature sensitive. Some resistance genes in cotton are known to be temperature sensitive [412].
Race-specific incompatibility may account for the varied X. c. pv. malvacearum (cotton strains) reactions on hibiscus. It was interesting to note that strains FL79 and TX84, both race 16 of X. c. pv. malvacearum of cotton strains, each differed in reactions on the hibiscus differential lines. Since Acala 44 elicited a hypersensitive response from 6 of 8 hibiscus strains, possibly Acala 44 contains an undiscovered resistance gene to the hibiscus derived strains. Those strains for which no hypersensitive reaction was induced may have escaped host recognition, but lack the necessary pathogenicity determinants to incite disease. Avirulence genes do not give phenotypic expression in non-parasites [234]. Bacterial growth kinetics would help determine if the bacteria are multiplying on these hosts. This would be useful for the case of X. c. pv. esculenti, for which 1 of 8 cotton hosts were incompatible, and 2 of 3 hibiscus hosts were incompatible.
The data from the protein and biochemical tests demonstrated differences between strains, but were not useful for typing strains by pathovar. There was significant variation among strains within a given pathovar. Although the use of PAGE to study total protein profiles did not differentiate the pathovars, some work has suggested limiting the analysis to just outer membrane proteins to better resolve the differences [333].
These experiments confirmed pathogenicity of the stock strains and clarified inconsistencies in the literature concerning host range. The host range studies indicated that conclusions reached may often be strain-dependent, and that examination of a large number of strains will reveal a different overall picture than an examination of a few strains. For example, the plant source of X. c. pv. malvacearum determines whether a strain will have the host range listed for the pathovar (hibiscus and cotton) or a more limited host range (hibiscus). However, the plant source of X. c. pv. alfalfae, does not determine whether a strain will have the host range listed. A strain which attacks both alfalfa and bean is X. c. pv. alfalfae. A strain with a host range limited to bean is X. c. pv. phaseoli. Finally, several biochemical methods were evaluated for their potential to classify a strain at the pathovar level. Although standard microbiological tests and protein gels were not adequate for such classifications, an examination of plasmid DNAs (Chapter 4) and chromosomal DNAs (Chapter 6) yielded more rewarding results.
Table 3-1. Strains of X. campestris used in host range investigation. Pathovar Strain Host Location Source ------------------------------------------------------------------------------------------------- alfalfae KS Medicago sativa Kansas D.L.Stuteville FL Medicago sativa Florida R.E.Stall cyamopsidis 13D5 Cyamopsis tetragonoloba - C.I.Kado X002,X005,X016,X017 Cyamopsis tetragonoloba Arizona J.Mihail glycines B-9-3 Glycine max Brazil W.F.Fett 1717 Glycine max Africa W.F.Fett 17915 Glycine max - W.F.Fett S-9-8 Glycine max Wisconsin W.F.Fett malvacearum D,M,N,O,U,V,X,Y,Z,TX84 Gossypium hirsutum Texas this study A,B,E,F,G,H Gossypium hirsutum Oklahoma M.Essenberg Ch1,Ch2 Gossypium hirsutum Chad L.S.Bird HV25 Gossypium hirsutum Upper Volta L.S.Bird Su2,Su3 Gossypium hirsutum Sudan L.S.Bird FL79 Gossypium hirsutum Florida this study 083-4244,M84-11 Hibiscus rosa-sinensis Florida DPI X10,X27,X52,X102,X108 Hibiscus rosa-sinensis Florida A.R.Chase phaseoli EK11,Xph25,Xpf11 Phaseolus vulgaris Nebraska M.Schuster Xpa,Xp11 Phaseolus vulgaris Wisconsin A.W.Saettler 82-1,82-2 Phaseolus vulgaris Florida R.E.Stall LB-2,SC-3B Phaseolus vulgaris Nebraska A.K.Vidaver XP2 Phaseolus vulgaris New York J.A.Laurence XP-JL Phaseolus vulgaris Kansas J.L.Leach XP-JF Phaseolus vulgaris Missouri this study XP-DPI,B5B Phaseolus vulgaris - this study pisi XP1 Pisum sativum Japan M.Goto vignicola A81-331,C-1,CB5-1, Vigna ungiuculata Georgia R.D.Gitaitis Xv19,SN2,432,82-38 Vigna unguiculata Georgia R.D.Gitaitis
Table 3-2. Legume plant reactions to inoculation with pathovars of X. campestris. Host ReactionA Pathovar phaseoli bean cowpea soybean alfalfa 82-1 +B -C -D 0E Xpa + - - 0 Xpf11 + - - 0 XP-JF + - - 0 EK11 + - - 0 XP2 + -F - 0 B5BG - - - 0 vignicola CB5-1 -H +I -J 0 Xv19 - + - 0 432 - + - 0 A81-331 - + - 0 C-1 - + - 0 82-38 +K + - 0 SN2 + + - 0 glycines B-9-3 +L -C +M 0 1717 + - + 0 17915 + - + 0 S-9-8G - - - 0 alfalfae FL +L -C -D +N malvacearum N -F 0 -F 0 control 0 0 0 0 ------------------------------------------------------- A = + is compatible, - is incompatible, + is intermediate, and 0 is a null reaction. B = compatible lesions were watersoaked and appeared to be spreading. C = dry necrotic lesion with wine red reaction. D = dry necrotic lesion with chlorosis. E = no reaction seen with spray inoculation. F = slight tissue discolorationat inoculation site. G = strain appeared non-pathogenic. H = dry necrotic lesion with slight watersoaking at periphery of inoculation site. I = dry necrotic lesion with shothole effect. J = dry necrotic lesion. K = as described for H, but slight shothole effect present. L = watersoaked lesion. M = watersoaked chlorotic lesion. N = watersoaked leaf spots.
Table 3-3. Host range observed for pathovars of Xanthomonas campestris pathogenic to legume host plants. Inoculation reaction on host plantsa Pathovar G.max M.sativa P.sativum P.vulgaris V.unguiculata alfalfaeb - + +c + -d glycines + - nt + - phaseoli -e - ntf + - pisi - - +c - - vignicola -e - nt +g + cyamopsidish - - nt - - a + = compatible, - = incompatible, 0 = null reaction, nt = not tested. Host plants were Glycines max (soybean), cultivar Evans; Medicago sativa (alfalfa), cultivar FL-77; Pisum sativum (pea), cultivar not known; Phaseolus vulgaris (bean), cultivar California Light Red; Vigna unguiculata (cowpea), cultivar California Blackeye #5. b other susceptible hosts reported are Trigonella and Melilotus. c reported as susceptible, but not tested. d reported as positive in the literature. e reaction appears negative for most strains, but occasional limited water soaking is evident. f there is a report of pathovar phaseoli on Pisum lunatus. g symptoms are similar to those seen in Vigna unguiculata. h the natural host belongs to the genus Cyamopsis.
Table 3-4. Malvaceous plant reactions to Inoculation with Strains of X. campestris pv. malvacearum. Cotton Hibiscus __________________Acala____________________ Strain 44 B1 B2 B3 B5 BIN 101 Gregg H1 H2 H3 ----------------------------------------------------------------------------------------------------------- N + + + + + + + + + 0 + H + - - - - - - - + 0 + FL79 + + + + + - - + + + + TX84 + + + + + - - + 0 0 0 X10 - - - - - - - - + + + X27 0 0 0 0 0 0 0 0 0 0 0 X52 - - - - - - - - - + + X102 - - - - - - - - - + + X103 - - - - - - - - - + + X108 - - - - - - - - + + + 83-4244 - - - - - - - - - + + M84-11 0 0 0 0 0 0 0 0 0 0 0 84-1093a 0 0 - 0 0 0 0 0 - + 0 ----------------------------------------------------------------------------------------------------------- a Strain 84-1093 is X. c. pv. esculenti.
Table 3-5. Biochemical reactions of strains of X. campestris. Pathovar starch gelatin cellulase N4.5 N7.0 N8.5 N5.0 Lecithin ---------------------------------------------------------------------------- alfalfae KS + + w - - - - + FL + + w - - - - + cyamopsidis 13D5 + + + - + + - + glycines B-9-3 + + + - + + - + 1717 + + + - + + - + 17915 + + + - + + - + S-9-8 + + + - + + - + malvacearum H + + + - - - - - N + + + - - - - - phaseoli EK11 + + - - - - - + Xph25 + + + - - - - + Xpf11 + + + - - - - - Xpa + w - - - - - + Xp11 + + - - - - - + 82-1 + + - - - - - + 82-2 + + - - - - - + LB-2 + + - - - - - + SC-3B + + + - + + - - XP2 + + - - - - - + XP-JL + + - - - - - + XP-JF + + - - - - - + XP-DPI + + - - - - - + pisi XP1 + + + - + + - + vignicola A81-331 + + + - + + - + C-1 + w + - + + - + CB5-1 + w + - + + - + Xv19 + w + - + + - + SN2 + w + - + + - + 432 + w + - + + - + 82-38 + w + - + + - + ---------------------------------------------------------------------------- all strains were mucoid, esculin positive, milk proteolytic, lipase positive. lecithinase positive reaction observed as a clearing zone plus a precipitation in the medium.
Figure 3-1. Polyacrylamide gel electrophoresis of total protein from pathovars of X. campestris.