One characteristic feature of many phytopathogenic organisms is their ability to produce an array of enzymes capable of degrading the complex polysaccharides of the plant cell wall (Bateman and Millar, 1966; Wood, 1967; Albersheim et al., 1969; Wood, 1973) and membrane constituents (Porter, 1966; Tseng and Bateman, 1968). These enzymes usually are produced inductively. Generally, they are extracellular, highly stable and present in infected host tissues. In most plant diseases caused by microbial agents, cell walls are penetrated, tissues are colonized, and permeability of host cells is altered. A brief summary of our understanding of cell wall and membrane structure, coupled with knowledge of the enzymes capable of degrading the components of these structures, and an analysis of the association of these enzymes with diseased tissue, should enable us to make an appraisal of their involvement in pathogenesis and point the way to an objective consideration of this area of disease physiology.

Degradation of Plant Cell Walls and Membranes by Microbial Enzymes

After harvest, vegetables are often spoiled by a wide variety of microorganisms including many bacterial and fungal species. The most common bacterial agents are Erwinia carotovora, Pseudomonas spp., Corynebacterium, Xanthomonas campestris, and lactic acid bacteria with E. carotovora being the most common, attacking virtually every vegetable type. Fungi commonly causing spoilage of fresh vegetables are Botrytis cinerea, various species of the genera Alternaria, Aspergillus, Cladosporium, Colletotrichum, Phomopsis, Fusarium, Penicillium, Phoma, Phytophthora, Pythium and Rhizopus spp., Botrytis cinerea, Ceratocystis fimbriata, Rhizoctonia solani, Sclerotinia sclerotiorum, and some mildews. A few of these organisms show a substrate preference whereas others such as Botrytis cinerea, Colletotrichum, Alternaria, Cladosporium, Phytophthora, and Rhizopus spp., affect a wide variety of vegetables causing devastating losses. Many of these agents enter the plant tissue through mechanical or chilling injuries, or after the skin barrier has been broken down by other organisms. Besides causing huge economic losses, some fungal species could produce toxic metabolites in the affected sites, constituting a potential health hazard for humans. Additionally, vegetables have often served as vehicles for pathogenic bacteria, viruses, and parasites and were implicated in many food borne illness outbreaks. In order to slow down vegetable spoilage and minimize the associated adverse health effects, great caution should be taken to follow strict hygiene, good agricultural practices (GAPs) and good manufacturing practices (GMPs) during cultivation, harvest, storage, transport, and marketing.

Spoilage of vegetable crops by bacteria and fungi and related health hazards

The phytopathogenic enterobacterium Erwinia chrysanthemi contains pel genes encoding several different isozymes of the plant-tissue-disintegrating enzyme pectate lyase (PL). The pelC gene, encoding an isozyme with an approximate isoelectric point of 8.0, was mutagenized by a three-step procedure involving (i) insertional inactivation of the cloned gene by ligation of a kan-containing BamHI fragment from pUC4K with a partial Sau3A digest of E. chrysanthemi pelC DNA in pBR322; (ii) mobilization of the pBR322 derivative from Escherichia coli to E. chrysanthemi by the helper plasmids R64drd11 and pLVC9; and (iii) exchange recombination of the pelC::kan mutation into the E. chrysanthemi chromosome by selection for kanamycin resistance in transconjugants cultured in phosphate-limited medium (which renders pBR322 unstable). The resulting E. chrysanthemi mutant was Kanr Amps, lacked pBR322 sequences, and was deficient in only one of the four major PL isozymes, PLc, as determined by activity-stained isoelectric-focusing polyacrylamide gels. The rates of PL induction and cell growth in a medium containing polygalacturonic acid as the sole carbon source were not significantly reduced in the mutant. No difference was detected in the ability of the mutant to macerate potato tuber tissue. The evidence suggests that this isozyme is not necessary for soft-rot pathogenesis.

Marker-exchange mutagenesis of a pectate lyase isozyme gene in Erwinia chrysanthemi.

Erwinia chrysanthemi produced extracellular enzymes which degrade pectic acid in a trans -eliminative manner. Isolate 307, from carnation, produced at least two, and isolate EC 23 , from Philodendron , produced at least four polygalacturonic acid trans -eliminases. The enzymes produced by each isolate were separated from each other and purified by ammonium sulfate fractionation, diethylaminoethylcellulose column chromatography, and electrofocusing. Molecular weights for the six enzymes ranged between 30,000 and 36,000 as determined by gel filtration in Sephadex G-75 and sucrose density gradient centrifugation. The isoelectric points of two enzyme fractions from isolate 307 were pH 9·1 and 7·9 and those for the four enzyme fractions from isolate EC 23 were pH 9·4, 8·4, 7·9 and 4·6. Random cleavage of pectic acid was greatest with the enzyme fractions having isoelectric points at pH 9·4 and 9·1, intermediate for the fraction with an isoelectric point at pH 4·6, and least with the fractions with isoelectric points at pH 8·4 and 7·9. All of the trans -eliminases exhibited a Ca 2+ requirement and had pH optima between pH 8·2 and 9·8. All but one of the purified polygalacturonic acid trans -eliminase fractions from both E. chrysanthemi isolates induced electrolyte loss, maceration and cell death of potato tissue. The enzyme fraction with an isoelectric point at pH 4·6 from isolate EC 23 did not macerate potato, carrot or cucumber tissue even though it readily degraded pectic acid prepared from onion, carrot and cucumber as well as commercial sources of sodium polypectate, polygalacturonic acid and pectin N.F.

Pectic enzymes produced by Erwinia chrysanthemi and their effects on plant tissue

The rhizosphere region is a variable zone containing a proliferation of microorganisms inside and outside the plant root. Many compounds are both taken up and passed out. Under normal growth conditions the rhizosphere exists because of the continuous loss of many forms of plant metabolites, which are rapidly utilized by microorganisms. Consequently, these rhizosphere microorganisms are in a position to affect both subsequent loss of material from the roots and nutrient uptake by the roots. In natural ecosystems an equilibrium develops between the plant and microorganisms that is affected only by the normal growth of plant and seasonal changes in the environment. However, in agriculture, man continually changes the normal equilibrium by manifold means. (e.g., plant monoculture, herbicide, fungicide and pesticide treatments, fertilizer application, and cultivation), all of which modify subsequent plant growth and the associated rhizosphere biota. Because of the importance of agriculture, the majority of work on the rhizosphere and its effects on plant growth has involved research on crop plants and, although this has provided great insight into rhizosphere—plant interactions in these relatively few species, some care should be taken in extrapolating such results to all natural ecosystems. With this proviso, we attempt to show, first, the effect the plant has on development and maintenance of the rhizosphere and, second, the influence the rhizosphere has on plant physiology and consequently crop productivity, highlighting areas of research likely to be rewarding both scientifically and commercially in the future. We do not attempt a complete review of the literature, since there have been reviews on many aspects of rhizosphere biology in recent years (Barber, 1978; Hale, et al. 1978; Newman, 1978; Balandreau and Knowles, 1978; Hale and Moore, 1979; Bowen, 1979, 1980, 1982; Woldendorp, 1981; Foster and Bowen, 1982; Lynch, 1982, 1983; Subba Rao, 1982a; Suslow, 1982), but rather choose specific examples to illustrate our major points.

M. S. Mount

Papers

Induction of electrolyte loss, tissue maceration, and cellular death of Potato tissue by an endopoly galacturonate trans-eliminase.