T.R. Swinburne

Production of antibiotics by Bacillus subtilis and their effect on fungal colonists of apple leaf scars

Apple rootstocks were sprayed after leaf fall with two antibiotic-yielding isolates of Bacillus subtilis. Both could be recovered from the leaf scar tissue throughout the dormant season and spring, but with time an increasing number of strains of B. subtilis were isolated, all of which possessed both antifungal and antibacterial activity similar to that of the original strains used. B. subtilis did not persist on the rootstock bark. The antifungal metabolites produced by B. subtilis were shown to be relatively stable compounds in vitro but the antibacterial metabolites were rapidly inactivated. The antifungal metabolites were found to be as inhibitory to many of the fungi commonly isolated from apple leaf scar tissue as they were to Nectria galligena.

Phytoalexin involvement in the latent infection of Capsicum annuum L. fruit by Glomerella cingulata (Stonem.)

Abstract Immature Capsicum annuum L. fruits wound-inoculated with conidia of Glomerella cingulata (Stonem.) did not develop progressive lesions until fruit ripening was well advanced. Juice expressed from inoculated tissue was inhibitory to G. cingulata. Thin layer chromatography (t.l.c.) of ether extracts of inoculated tissue, bioassayed with G. cingulata or Cladosporium, demonstrated the presence of one prominent inhibition zone. This fungitoxic compound, capsicannol, began to accumulate within 18 h after inoculation in the immature fruit tissue, reached maximum concentrations after 4 days and was more concentrated in superficial than in deeper tissues. Capsicannol and capsidiol, which accumulated in inoculated ripening fruit tissue, had declined to non-toxic levels at the onset of progressive lesion development. Diminution of the 2 phytoalexins was accompanied by the appearance of capsenone and another compound which showed slight fungitoxicity. Capsidiol and capsicannol, which accumulated in tissue treated with an elicitor from G. cingulata mycelial walls, declined at a rate similar to that in tissue inoculated with conidia, and both compounds were degraded in liquid culture by G. cingulata.

Benzoic acid: an antifungal compound formed in Bramley's Seedling apple fruits following infection by Nectria galligena Bres

Abstract The resistance of immature apples of the variety Bramleys Seedling to rotting by Nectria galligena was found to be associated with the production of an antifungal compound after infection. The active compound was purified and identified as benzoic acid which was shown to be toxic only in acid conditions. Sufficient quantities were found in the diseased tissue to account for all the antifungal activity present at the pH of the tissue.

Iron-chelating agents and lesion development by Botrytis cinerea on leaves of Vicia faba

Abstract Botrytis cinerea produced lesions on leaves of Vicia faba more rapidly and many more spreading lesions develope when inocula contained the iron chelating agents EDTA or DHBA than when conidia were inoculated in water alone. Treatment of conidia with EDTA for 3 h prior to inoculation also resulted in increased lesion development. Fe-EDTA or Fe-DHBA in inoculum drops reduced lesion formation to a very low level and no spreading lesions developed. EDTA and DHBA had little effect on germination of conidia on glass slides but were slightly stimulatory to appressoria formation. Fe-EDTA and Fe-DHBA reduced the percentage germination to below 5% and no appressoria were produced. At the leaf surface, however, both germination and appressoria formation were stimulated by EDTA and DHBA whereas Fe-EDTA and Fe-DHBA reduced the percentage germination and appressoria formation when compared with the water controls. Phytoalexins accumulated in the restricted lesions caused by B. cinerea in water but only low levels accumulated in the spreading lesions caused by B. cinerea in EDTA or DHBA solution and at the site of inoculation with conidia in Fe-EDTA or Fe-DHBA solution which remained symptomless. The importance of iron in the aggressiveness of B. cinerea infecting V. faba, and of 2 other pathogens on their host, is discussed.

Observations on infection of Capsicum annuum fruit by Glomerella cingulata and Colletotrichum capsici

Conidia of G. cingulata and C. capsici germinated at a similar rate on the surface of immature, ripening and fully ripened C. annuum fruits. G. cingulata produced appressoria more rapidly on the surface of immature fruits than on ripening or fully ripened fruits; a few appressoria produced infection hyphae which penetrated the cuticle and outer epidermal cell wall of immature fruits then ceased further growth, but most appressoria remained dormant. Less than 40% of germinated C. capsici conidia produced appressoria at the surface of fruits at any stage of ripeness and none developed in water or in leachates on glass slides. The appressoria on the surfaces of immature fruits did not develop infection hyphae and remained dormant. Both G. cingulata and C. capsici remained quiescent on immature fruits and developed progressive lesions only when the fruits had fully ripened. Neither fungus developed many progressive lesions on intact fruits, even when fully ripened, but in wound-inoculated fruits C. capsici developed progressive lesions several days earlier than G. cingulata and required a smaller inoculum to do so. Immature fruits inoculated with G. cingulata and C. capsici accumulated similar quantities of the phytoalexin, capsicannol, but C. capsici was shown to be slightly less sensitive than G. cingulata to capsicannol.

A Role for Iron in Germination of Conidia of Colletotrichum musae

The time course of germination of conidia of Colletotrichum musae in the presence of different germination stimulants is consistent with the postulate that the stimulation of germination by anthranilic acid can be attributed to its previously established conversion to 2,3-dihydroxybenzoic acid in conidia. The activity of a number of synthetic chelating agents, such as EDTA and 1,10-phenanthroline, in promoting germination of the same order as 2,3-dihydroxybenzoic acid at about the same molarity provides support for the hypothesis that the chelating properties of the latter compound are important in its biological action. Experiments with conidia produced on media of different iron concentrations indicate that in the absence of a germination stimulant there is a high inverse correlation between the percentage germination of conidia and conidial iron content. However, in the presence of anthranilic acid there is no significant correlation between these parameters, percentage germination remaining uniformly high regardless of the conidial iron concentration. It is concluded that conidial iron is involved in the inhibition of germination and that this inhibition can be relieved non-specifically by a variety of chelating agents.

Relationship of spore germination and appressoria formation in isolates of Septoria nodorum to pathogenicity on wheat and barley leaves

A wheat isolate (WI 1 ) and one barley isolate (BI 1 ) of Septoria nodorum showed little change in pathogenicity after one passage through the opposite host, but after two or three passages some loss in pathogenicity to the original host was observed. Some re-isolates of a second barley isolate (BI 2 ) showed morphological characteristics typical of wheat isolates after a single passage through wheat, and after a second passage through wheat a morphologically changed barley re-isolate was as pathogenic on wheat as the original wheat isolate and showed poor pathogenicity on barley; repeated passage back through barley failed to reverse this effect. Spores of WI 1 germinated to a similar extent in both wheat and barley leaf leachates, as did BI 2 , but spores of WI 1 tended to germinate more rapidly. The proportion of germinated spores of WI 1 and BI 2 producing appressoria was, however, greater in leachates from wheat leaves. Germination in both isolates was similar on the surface of wheat and barley leaves during the first 6 h incubation, although after 24 h incubation a higher percentage germination was observed on the original host. Hyphae of WI 1 were considerably more branched on wheat leaves than on barley leaves while hyphae of BI 2 were more branched on barley leaves. Hyphae of the morphologically changed barley re-isolate (BI 2 ) were more branched on wheat leaves. Appressoria were produced at the tips of most hyphal branches of all isolates.

Influence of iron and iron chelators on formation of progressive lesions by Colletotrichum musae on banana fruits

Conidia from Colletotrichum musae cultures grown in iron-deficient medium (Fe3+ Cell-wall glucans extracted from C. musae hyphae grown in iron-deficient medium elicited browning and the accumulation of the same antifungal compounds as the elicitor from hyphae grown in iron-replete medium. The addition of the iron-chelating agents to the elicitors did not inhibit the formation of the antifungal compounds.

2,3-Dihydroxybenzoic acid and related compounds as stimulants of germination of conidia of Colletotrichum musae (Berk. & Curt) Arx

Abstract Anthranilic acid, a stimulant of germination in leachates of banana fruits, was converted in replacement culture experiments to 2,3-dihydroxybenzoic acid, catechol and pyrogallol by mycelium of Colletotrichum musae. 3-Hydroxyanthranilic acid was not an intermediate in the formation of 2,3-dihydroxybenzoic acid. Both catechol and pyrogallol were produced when mycelium was incubated with 2,3-dihydroxybenzoic acid, but incubation of mycelium with catechol did not yield pyrogallol, suggesting that both of these compounds were derived from 2,3-dihydroxybenzoic acid. The same catabolites were detected when conidia were incubated with anthranilic acid. Concentrations of 2,3-dihydroxybenzoic acid and catechol were maximal after 8 h incubation, when under the conditions of the experiment approximately one-third of the conidia had germinated, and declined thereafter. In bioassay experiments the concentration of 2,3-dihydroxybenzoic acid required to stimulate 50% germination was significantly less (2·5 × 10−5 m ) than that of anthranilic acid (3·7 × 10−4 m ) or catechol (1·3 x 10−3 m ). Pyrogallol was not effective in the assay. The possibility that the mode of action of these and other compounds involves their chelating properties is discussed.

Host range of Nectria galligena Bres. and the pathogenicity of some Northern Ireland isolates

This paper reviews the recorded host range of Nectria galligena Bres. and describes cross-inoculation experiments with N. galligena, N. ditissima and N. coccinea isolated from various host species, particularly those found near apple plantations. N. galligena isolated from typical apple cankers induced lesions on poplar, hawthorn and beech but not ash or sycamore. Isolates from lesions on ash induced cankers on apple, hawthorn, poplar and beech but the symptoms on apple were atypical. The incitant of ash canker was therefore considered to be a forma specialis, N. galligena f.sp. fraxini distinct from the incitant of apple canker, N. galligena f.sp. mali . Both were recovered from natural cankers on hawthorn. N. galligena f.sp. mali and N. ditissima were isolated from beech but the latter was pathogenic only to beech. N. coccinea isolated from sycamore was pathogenic only to sycamore and hawthorn. Poplar, hawthorn and to a lesser extent beech were the only external sources of inoculum detected for infection of apple plantations. Windbreaks of grey alder were not found infected under natural conditions, and although cankers developed following inoculation with N. galligena these healed within l or 2 years.