Edo Chalutz

Mode of action of the postharvest biocontrol yeast, Pichia guilliermondii. I. Characterization of attachment to Botrytis cinerea

Abstract An isolate (87) of the yeast Pichia guilliermondii, protects apples from postharvest fruit rotting fungi Botrytis cinerea and Penicillium expansum. In order to examine the yeast-pathogen interaction, B. cinerea was grown on agar plates overlayed with cellophane. Effective and non-effective yeast isolates were applied near the young hyphal growth. Samples were taken 24 h later from the area where the fungi and yeast had intersected. Light microscopy revealed a general attachment of the effective biocontrol agent P. guilliermondii (isolate 87) and a non-effective isolate (117) of Debaryomyces hansenii. Low temperature scanning electron microscopy (LTSEM) indicated that both species of yeast attached to the fungal hyphae, but the 87 isolate attached fastidiously. Twenty-four hours after applying the 87 isolate to B. cinerea, pitting and collapse of the hyphae were observed. These observations were confirmed using transmission electron microscopy. These features were not observed with the ineffective isolate of D. hansenii. Further experiments indicated that attachment of P. guilliermondii to hyphae of B. cinerea could be blocked by agents that alter protein integrity (salts, proteases, etc.) and certain sugars. Isolates of both species produced β-(1–3) glucanase when cultured in various carbon sources and on cell walls of fruit rotting pathogens. Culture supernatants from P. guilliermondii, however, yielded two- to five-fold more β-(1–3) glucanase activity compared with D. hansenii. Data indicate that tenacious attachment, along with secretion of cell wall degrading enzymes, may play a role in the biocontrol activity of this yeast antagonist.

A selection strategy for microbial antagonists to control postharvest diseases of fruits and vegetables

Abstract Fruit wounds were utilized to screen for potential antagonists to postharvest rot organisms from unidentified microbial populations on fruit surfaces. Washings from apple, oranges, and tomato fruit surfaces were placed in fresh apple fruit wounds which were subsequently challenged with postharvest rot pathogens. After 10 days, those wounds not showing rot development were scraped with a sterile needle. Dislodged material was diluted in sterile water which was diluted serially and plated on nutrient agar. Individual isolations were made from frequently occurring colonies and tested for antagonistic activity against postharvest pathogens. Utilizing these procedures, six isolates were identified which were effective antagonists against Botrytis and Penicillium rots of apple. Three of the isolates were identified as Candida sake (Saito & Ota) Van Uden & Buckley and one as Candida tenuis Diddens & Lodder none of which has been described previously as a biocontrol agent.

Postharvest biological control of Penicillium rots of citrus with antagonistic yeasts and bacteria

Abstract Two yeasts, Debaryomyces hansenii (Zopf) Van Rij and Aureobasidium pullulans (De Bary) Arnaud, and two bacteria, Pseudomonas cepacia (Van Hall) Bergy et al. and P. syringae Burkholder, were the most effective antagonists from over 100 isolates tested against Penicillium digitatum Sacc. and Penicillium italicum Wehmer rots on citrus fruit. Overall, P. cepacia provided the best protection against both of these rots. P. cepacia produced antibiotic zones against the two Penicillia rot organisms in culture, whereas P. syringae, D. hansenii and A. pullulans did not. All four antagonists show promise as biocontrol agents.

Biological control of post-harvest diseases of fruits and vegetables: alternatives to synthetic fungicides

Spoilage of fruits and vegetables after harvest often causes losses as great as 25-50% of the harvested crop. Much of this is due to rot micro-organisms which are currently controlled by refrigeration and fungicides. A number of bacterial and fungal antagonists have been found that can effectively control post-harvest rots of peaches, citrus, apples, grapes and tomatoes. These antagonists have various modes of action that include antibiosis and/or competition for nutrients and space. The commercialization of some of these antagonists to control post-harvest decay of fruits and vegetables appears to be feasible and may present an alternative to synthetic pesticides.

The expression of a grapefruit gene encoding an isoflavone reductase-like protein is induced in response to UV irradiation

Exposure of harvested grapefruit to UV-C (254 nm) irradiation was previously found to induce resistance against the green mold decay caused by Penicillium digitatum. In order to gain insight into the mechanism of this UV-induced resistance we initiated a study for isolation of genes induced during this process. Using the differential display method we cloned cDNA representing an mRNA which is accumulated in grapefruit peel upon UV irradiation. Sequence analysis revealed that this cDNA represents a gene encoding for an isoflavone reductase-like protein and was termed IRL (isoflavone reductase-like). The grapefruit IRL protein sequence has high homology also to a novel family of other isoflavone reductase-like proteins present in few non-legume plants and whose function is not clear yet. The UV dose, and time following it, which lead to maximal accumulation of the IRL transcript were found to be similar to those leading to maximal induced resistance. The expression of the IRL gene was demonstrated to be induced also by wounding and pathogen infection.

Fungal infections suppress ethylene-induced phenylalanine ammonia-lyase activity in grapefruits

Ethylene-induced phenylalanine ammonia-lyase (PAL) activity in yellow grapefruit peel was inhibited by inoculation of the fruit with Penicillium digitatum , not only at a place close to the infection site but also in areas farther away from it. The degree of inhibition was related to the distance of the fungus from the assayed region. Other fungi tested such as a non-ethylene producing isolate of P. digitatum , an isolate of P. italicum and an isolate of Geotrichum candidum had a similar effect. This phenomenon was also found in orange fruit peel. PAL was not induced in the green fruit by ethylene and also not induced during natural infection of mature fruits by P. digitatum in spite of the production of ethylene during infection. These findings indicate a mechanism whereby fungi are able to prevent the defence response of host cells to invasion.

The contribution of host and pathogen to ethylene biosynthesis in Penicillium digitatum-infected citrus fruit

Abstract The biosynthesis of ethylene by grapefruit ( Citrus paradisi Macf) cv. “Marsh Seedless” 6 days after inoculation with either an ethylene producing or a non-ethylene-producing isolate of Penicillium digitatum , was studied. Relatively low levels of ethylene were produced by the apparently healthy region, at the edge of rot in fruits infected with either the ethylene producing or the nonethylene-producing isolate of the fungus. Higher levels of production were found in regions of the peel showing infection symptoms, but only in fruit infected with the ethylene-producing isolate. The production of symptoms and the ACC content of the peel of fruits inoculated with the ethylene-producing isolate was similar to that in fruits inoculated with the non-producing isolate. The use of radiolabelled precursors showed that the ethylene produced by the healthy parts of the fruit originated from methionine, while the ethylene produced by the infected parts of the fruit originated mostly from glutamic acid. Furthermore, ethylene production by the healthy part of the fruit was markedly enhanced by 1-aminocyclopropane-1-carboxylic acid (ACC), and to a lesser extent by CuSO 4 , but it was inhibited by aminoethoxyvinylglycine (AVG). In contrast the production of ethylene by infected peel and by P. digitatum in culture was not affected by ACC, but was markedly inhibited by CuSO 4 , and, to a lesser extent, by AVG. These results suggest that ethylene production in the healthy part of the fruit is of plant origin, whereas the markedly enhanced production of ethylene by the P. digitatum -infected regions, is mostly or entirely offungal origin.

Ethylene biosynthesis and related physiological changes in Penicillium digitatum-infected grapefruit (Citrus paradisi)

Abstract Biochemical and physiological changes and their relation to ethylene biosynthesis were studied in grapefruit ( Citrus paradisi Macf. cv. Marsh Seedless) peel, 5–6 days after inoculation with Penicillium digitatum Sacc. In both the albedo and flavedo tissues of the peel, fungal invasion was associated with increases in free galacturonic acid but with reductions in pH and soluble proteins. The extent of the changes was smaller the greater the distance from the maceration front. Two parallel and distinct maceration fronts could be defined in the peel, the one in the albedo preceding that in the flavedo. Fungal glucosamine was present in the apparently healthy region of the albedo up to 15 mm ahead of the flavedo maceration front. Fungal invasion was associated with increases in both 1-aminocycloproprane-1-carboxylic acid (ACC) and ethylene production but the ability of the tissue to convert ACC to ethylene decreased with the development of the infection. The early relatively low rate of ethylene production in infected fruit seems to originate mostly from the fruit tissue while a later and higher rate of ethylene production originates mostly from the fungus.

No rôle for ethylene in the pathogenicity of Penicillium digitatum

Abstract A non-ethylene-producing isolate of the green mould fungus Penicillium digitatum Sacc. was used to test the role of ethylene in the pathogenicity of this fungus on citrus fruits. This isolate was similar to the wild-type, ethylene-producing isolate in morphological characteristics and growth rate both in vitro and in vivo . However, it did not produce ethylene in vitro, nor was ethylene produced by the disease caused by this isolate. Based on these finidings, it was concluded that ethylene, which is normally produced at very high rates by Penicillium digitatum Sacc. in vitro or in vivo , has no clear role in the pathogenicity of this fungus.

Purification and Properties of the Ethylene-Inducing Factor from the Cell Wall Digesting Mixture, Cellulysin

The production of ethylene during development and senescence of higher plants is a well established fact. Also, stress (including physical and chemical wounding), or application of herbicides and hormones, as well as host-pathogen interaction induce ethylene production (11). Recently we reported a new type of ethylene-inducing agent, a cell wall digesting enzyme mixture, ‘Cel1ulysin’ (4). The induction of ethylene in tobacco leaves (4) by Cellulysin can be relatively fast, within 30 minutes, compared to that by the hormope IAA which takes hours (2). Cellulysin-mediated induction of ethylene biosynthesis is inferred to be at the level of ACC synthase because ACC is produced and 0.lmM aminoethoxyvinylglycine (AVG), a known inhibitor of this enzyme (1), inhibits both the induction of ACC and ethylene biosynthesis. The mechanism by which Cellulysin induces ethylene biosynthesis is not known, but it occurs even in the presence of the protein synthesis inhibitors, cycloheximide (50mM) and chloramphenicol (100 µg/ml).