Lipopeptides, a novel protein, and volatile compounds contribute to the antifungal activity of the biocontrol agent Bacillus atrophaeus CAB-1

Bacillus atrophaeus CAB-1 displays a high inhibitory activity against various fungal pathogens and suppresses cucumber powdery mildew and tomato gray mold. We extracted and identified lipopeptides and secreted proteins and volatile compounds produced by strain CAB-1 to investigate the mechanisms involved in its biocontrol performance. In vitro assays indicated all three types of products contributed to the antagonistic activity against the fungal pathogen Botrytis cinerea. Each of these components also effectively prevented the occurrence of the cucumber powdery mildew caused by Sphaerotheca fuliginea under greenhouse conditions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry revealed that the major bioactive lipopeptide was fengycin A (C15–C17). We isolated the crude-secreted proteins of CAB-1 and purified a fraction with antifungal activity. This protein sequence shared a high identity with a putative phage-related pre-neck appendage protein, which has not been reported as an antifungal factor. The volatile compounds produced by CAB-1 were complex, including a range of alcohols, phenols, amines, and alkane amides. O-anisaldehyde represented one of the most abundant volatiles with the highest inhibition on the mycelial growth of B. cinerea. To our knowledge, this is the first report on profiling three types of antifungal substances in Bacilli and demonstrating their contributions to plant disease control.     

Expression of Paenibacillus polymyxa β-1,3-1,4-glucanase in Streptomyces lydicus A01 improves its biocontrol effect against Botrytis cinerea

Streptomyces lydicus strain A01, which can produce natamycin and chitinase, has a significant inhibition effect on gray mold disease caused by Botrytis cinerea. However, it has no detectable glucanase activity. Strain A21 isolated from the snow covered high altitude area in Tibet, China, also has a high antagonistic activity against B. cinerea. It displayed an obvious halo on lichen polysaccharides plates by congo red staining, indicating a strong glucanase activity. A21 was identified as Paenibacillus polymyxa using 16S rDNA gene analysis and biochemical and physiological analysis. To obtain the synergistic antifungal effects of natamycin, chitinase, and glucanases on B. cinerea, this study transformed the β-1,3-1,4-glucanase gene from P. polymyxa A21 to S. lydicus A01. The engineered S. lydicus AG01 showed substantially high glucanase activity, and had similar natamycin production and chitinase activity as the wild-type strain A01. Compared to the wild-type strain A01, the antifungal effects of S. lydicus AG01 on B. cinerea, including inhibition of spore germination and mycelial growth, were highly improved. The improved biocontrol effect of S. lydicus AG01 is likely attributed to the heterologous expression of glucanase from P. polymyxa, which acted synergistically with natamycin and chitinase to increase the antifungal activity of the strain.     

Characterization of Genes Involved in Biosynthesis of a Novel Antibiotic from Burkholderia cepacia BC11 and Their Role in Biological Control of Rhizoctonia solani

Genetic manipulation of fluorescent pseudomonads has provided major insight into their production of antifungal molecules and their role in biological control of plant disease. Burkholderia cepacia also produces antifungal activities, but its biological control activity is much less well characterized, in part due to difficulties in applying genetic tools. Here we report genetic and biochemical characterization of a soil isolate of B. cepacia relating to its production of an unusual antibiotic that is very active against a variety of soil fungi. Purification and preliminary structural analyses suggest that this antibiotic (called AFC-BC11) is a novel lipopeptide associated largely with the cell membrane. Analysis of conditions for optimal production of AFC-BC11 indicated stringent environmental regulation of its synthesis. Furthermore, we show that production of AFC-BC11 is largely responsible for the ability of B. cepacia BC11 to effectively control the damping-off of cotton caused by the fungal pathogen Rhizoctonia solani in a gnotobiotic system. Using Tn5 mutagenesis, we identified, cloned, and characterized a region of the genome of strain BC11 that is required for production of this antifungal metabolite. DNA sequence analysis suggested that this region encodes proteins directly involved in the production of a nonribosomally synthesized lipopeptide.     

Identification of Botryticidal Proteins with Similarity to NBS–LRR Proteins in Rosemary Pepper (<Emphasis Type="Italic">Lippia sidoides</Emphasis> Cham.) Flowers

Heavy agricultural losses are closely related to attacks by insect-pests and phytopathogens such as bacteria and fungi. Among them, the fungus Botrytis cinerea can cause gray mold in more than 200 different species of plants, and is considered a challenging problem for agribusiness. Fungicides are commonly used to control this pathogen because they are fast-working and easy to apply. However, the continuous use of fungicides may promote the selection of resistant fungi and can also cause profound contamination in ecosystems. Aiming to find alternative strategies to solve these problems, several studies have focused on searching for plant proteins and peptides with antifungal activities (AFPs). With this in mind, this report shows the isolation and characterization of two novels antifungal proteins from flowers of rosemary pepper (Lippia sidoides Cham.) with 10 and 15 kDa. Isolation was performed by using an Octyl-Sepharose hydrophobic column. In vitro bioassays indicated that isolated proteins were able to inhibit B. cinerea development, but were not effective against all bacteria tested. Moreover, N-termini sequences indicate that both proteins showed sequence homology with NBS–LRR R proteins with a lower molecular mass, suggesting possible protein fragmentation. Data reported here could help in the development of biotechnological products for crop protection against phytopathogenic fungi in the near future.     

Antifungal activity of zinc oxide nanoparticles against Botrytis cinerea and Penicillium expansum

Antifungal activities of zinc oxide nanoparticles (ZnO NPs) and their mode of action against two postharvest pathogenic fungi (Botrytis cinerea and Penicillium expansum) were investigated in this study. ZnO NPs with sizes of 70 ± 15 nm and concentrations of 0, 3, 6 and 12 mmol l⁻¹ were used. Traditional microbiological plating, scanning electron microscopy (SEM), and Raman spectroscopy were used to study antifungal activities of ZnO NPs and to characterize the changes in morphology and cellular compositions of fungal hyphae treated with ZnO NPs. Results show that ZnO NPs at concentrations greater than 3 mmol l⁻¹ can significantly inhibit the growth of B. cinerea and P. expansum. P. expansum was more sensitive to the treatment with ZnO NPs than B. cinerea. SEM images and Raman spectra indicate two different antifungal activities of ZnO NPs against B. cinerea and P. expansum. ZnO NPs inhibited the growth of B. cinerea by affecting cellular functions, which caused deformation in fungal hyphae. In comparison, ZnO NPs prevented the development of conidiophores and conidia of P. expansum, which eventually led to the death of fungal hyphae. These results suggest that ZnO NPs could be used as an effective fungicide in agricultural and food safety applications.     

Design,synthesis and antifungal activity evaluation of isocryptolepine derivatives

In this paper, the nitrogen atom was inserted into the anthracycline system of the isocryptolepine nucleus to obtain the “Aza”-type structure benzo[4,5]imidazo[1,2-c] quinazoline. A series of “Aza”-type derivatives were designed, synthesized and evaluated for their antifungal activity against six plant fungi in vitro. Among all derivatives, compounds A-0, B-1 and B-2 showed significant antifungal activity against B. cinerea with the EC₅₀ values of 2.72 μg/mL, 5.90 μg/mL and 4.00 μg/mL, respectively. Compound A-2 had the highest activity against M. oryzae with the EC₅₀ values of 8.81 μg/mL, and compound A-1 demonstrated the most control efficacy against R. solani (EC₅₀, 6.27 μg/mL). Moreover, compound A-0 was selected to investigate the in vivo tests against B. cinerea and the results indicated that the preventative efficacy of it up to 72.80% at 100 μg/mL. Preliminary mechanism studies revealed that after treatment with A-0 at 5 µg/mL, the B. cinerea mycelia appeared curved, collapsed and the cell membrane integrity may be damaged. The reactive oxygen species production, mitochondrial membrane potential and nuclear morphometry of mycelia have been changed, and the membrane function and cell proliferation of mycelia were destroyed. Compounds A-0, A-1, B-1 and B-2 presented weaker toxicities against two cells lines than isocryptolepine. This study lays the foundation for the future development of isocryptolepine derivatives as environmentally friendly and safe agricultural fungicides.     

Pyrrolnitrin Production by Biological Control Agent Pseudomonas cepacia B37w in Culture and in Colonized Wounds of Potatoes

Bacterial strain B37w (= NRRL B-14858), an isolate noteworthy because it inhibits the growth of the bioherbicide fungus Colletotrichum truncatum, was selected for further studies of bacterial antifungal properties. This isolate was identified as a Pseudomonas cepacia strain by performing carbohydrate utilization and fatty acid profile analyses, as well as other biochemical and physiological tests. Petri plate assays revealed that strain B37w exhibited antifungal activity against the potato dry rot fungus Fusarium sambucinum. Using bioautography, we correlated antifungal activity with production of a specific compound. Isolation from strain B37w and identification of the antifungal antibiotic pyrrolnitrin are described. A whole-potato assay revealed B37w's ability to colonize potato wounds. Wounded potatoes were inoculated with B37w, and pyrrolnitrin was detected in these potatoes by thin-layer chromatography-bioautography at a concentration on the order of nanograms per wound. We performed an assay in which we examined efficacy against F. sambucinum-incited potato dry rot and found that B37w inhibited disease development. This is the first report of P. cepacia or pyrrolnitrin activity against the economically important potato pathogen F. sambucinum.     

Purification and characterization of antifungal compounds from Bacillus coagulans TQ33 isolated from skimmed milk powder

Bacillus coagulans TQ33, isolated from skimmed milk powder, displays strong antifungal activity against plant pathogenic fungi. The antifungal compound of the B. coagulans TQ33 culture was extracted by thin-layer chromatography and column chromatography, and its structure was elucidated based on HPLC, LC-MS, and NMR analysie. The antifungal compound was identified as phenyllactic acid (PLA), and it was found to have a minimum inhibitory concentration on Phytophthora drechsleri Tucker of 18 mg/mL. Bio-control activity tests indicated that PLA has a wide spectrum of antagonistic effects against Fusarium oxysporum, Botrytis cinerea, Glomerella cingulata, Penicillium citrinum, Penicillium digitatum, particularly against F. oxysporum. PLA is the most notable antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi that has been isolated and identified to date. These results indicate that B. coagulans TQ33 has the potential for application in biological pesticides.     

Effect of extracts from in vitro-grown shoots of <Emphasis Type="Italic">Quillaja saponaria</Emphasis> Mol. on <Emphasis Type="Italic">Botrytis cinerea</Emphasis> Pers.

The ethanolic and aqueous extracts from in vitro shoots of Quillaja saponaria Mol. (Quillay) were studied for their antifungal activity against the phytopathogenic fungus Botrytis cinerea Pers. These extracts reduced conidial germination and mycelial growth of B. cinerea, ethanolic extracts being more active than aqueous extracts. In addition, the damage areas produced by this fungus on tomato leaves and strawberry fruits pre-treated with quillay extracts were diminished. The fungitoxic effect of in vitro-grown quillay extract was similar to those obtained with commercial fungicides of both natural (BC-1000) and synthetic (iprodione–dicarboximide) origin. On the other hand, the antifungal action of quillay extracts obtained from adult trees naturally grown was only slightly superior to the fungitoxic activity of the extract from in vitro plants. HPLC analysis of the extract showed that it contained saponins and some phenolic compounds such as chlorogenic, caffeic, vanillic, and salicylic acids, and scopoletin, which have been identified as antifungal agents on phytopathogenic fungi. The results obtained in this work, suggests that extracts of in vitro-grown quillay have an important protective effect against B. cinerea and support the use of an in vitro culture system as a biotechnological alternative to obtain environmental safe antifungal quillay extracts to control B. cinerea, contributing to the preservation of this indigenous Chilean species.     

Pseudozyma aphidis activates reactive oxygen species production,programmed cell death and morphological alterations in the necrotrophic fungus Botrytis cinerea

Many types of yeast have been studied in the last few years as potential biocontrol agents against different phytopathogenic fungi. Their ability to control plant diseases is mainly through combined modes of action. Among them, antibiosis, competition for nutrients and niches, induction of systemic resistance in plants and mycoparasitism have been the most studied. In previous work, we have established that the epiphytic yeast Pseudozyma aphidis inhibits Botrytis cinerea through induced resistance and antibiosis. Here, we demonstrate that P. aphidis adheres to B. cinerea hyphae and competes with them for nutrients. We further show that the secreted antifungal compounds activate the production of reactive oxygen species and programmed cell death in B. cinerea mycelium. Finally, P. aphidis and its secreted compounds negatively affect B. cinerea hyphae, leading to morphological alterations, including hyphal curliness, vacuolization and branching, which presumably affects the colonization ability and infectivity of B. cinerea. This study demonstrates additional modes of action for P. aphidis and its antifungal compounds against the plant pathogen B. cinerea.     

Biotransformation of geranylated- and acetylated-phloroglucinols by Gibberella fujikuroi into molecules with increased antifungal activity against Botrytis cinerea

Terpenylated phenols possess interesting biological activities. These properties vary mainly according to the type of terpene associated and the degree of oxidation of the molecule. The search for new active molecules for application in different areas of knowledge includes the structural modification of these through ecological methodologies, such as biotransformation. The aims of this study were the biotransformation of geranylated- and acetylated-phloroglucinol by the fungus Gibberella fujikuroi and the evaluation of the antifungal activity of the derivatives. Five major derivatives were identified after biotransformation, highlighting the formation of specific monoacetylated products. In vitro antifungal activity assays against the phytopathogenic fungus Botrytis cinerea indicated that deacetylated derivatives possess higher activity compared to the precursor molecule. In other biotransformation reactions, a relationship between the release of the alkyl chain from the aromatic ring with a decrease of the antifungal activity, was observed. The in vivo tests in infected tomato plants with B. cinerea confirmed the antifungal activity of the derivatives observed in in vitro experiments.     

Development of novel 2-substituted acylaminoethylsulfonamide derivatives as fungicides against Botrytis cinerea

Botrytis cinerea is an economically important fungal pathogen with a host range of over 200 plant species. Unfortunately, gray mold disease caused by B. cinerea has not been effectively controlled because of its high risk for fungicide resistance development. As a part of our ongoing efforts to develop novel sulfonamides as agricultural fungicides against Botrytis cinerea, we introduced 2-aminoethanesulfonic acid (taurine) substructure, designed and synthesized a series of novel 2-substituted acylaminoethylsulfonamides. The newly synthesized sulfonamides were evaluated in vitro and in vivo for their fungicidal activity against Botrytis cinerea, of which the 2-ethoxyacetylamide derivative (V-A-12, EC₅₀ = 0.66 mg·L⁻¹) exhibited the highest potency in vitro and superior fungicidal activity compared with procymidone (EC₅₀ = 1.06 mg·L⁻¹). In vivo bioassay indicated that compound V-A-12 could be effective for the control of tomato gray mold. Moreover, the structure-activity relationship of these sulfonamides was analyzed by establishing a three-dimensional quantitative structure-activity relationship (3D-QSAR) model, which can provide guidance for the development of sulfonamides as fungicides. Finally, the effeicacy of sulfonamide derivatives was again verified in the activity evaluation against resistant Botrytis cinerea strains. These results further enhance the development value of 2-substituted acylaminoethylsulfonamides to control the tomato gray mold.     

Characterization of new bacterial biocontrol agents Acinetobacter,Bacillus, Pantoea and Pseudomonas spp. mediating grapevine resistance against Botrytis cinerea

A collection of 282 bacterial isolates from the rhizosphere and different organs of healthy field-grown grapevine plants was obtained and screened for their ability to protect grapevine leaves against Botrytis cinerea, the causal agent of gray mold. Twenty-six strains effectively controlled B. cinerea infections on leaves. After phenotypic and molecular analysis, seven strains were identified as Pseudomonas fluorescens PTA-268 and PTA-CT2, Bacillus subtilis PTA-271, Pantoea agglomerans PTA-AF1 and PTA-AF2, and Acinetobacter lwoffii PTA-113 and PTA-152. In vitro antifungal experiments showed that from these seven strains, only PTA-AF1 and PTA-CT2 exhibited a direct antagonism against B. cinerea. Furthermore, the biocontrol activity of the seven bacteria was associated with differential induction of defense-related responses lipoxygenase, phenylalanine ammonia-lyase and chitinase in grapevine leaves. Our results show that the selected bacteria can efficiently protect grapevine leaves against gray mold disease through an induction of plant resistance and in some cases by an additional antagonistic activity.     

Diversity and biocontrol potential of endophytic fungi in Brassica napus

This study was conducted to isolate endophytic fungi from oilseed rape (Brassica napus), to identify the fungal endophytes based on morphology and ITS (ITS1-5.8S rDNA-ITS2) sequences, and to evaluate their efficacy in suppression of the plant pathogenic fungi Sclerotinia sclerotiorum and Botrytis cinerea. Selected endophytic fungal isolates were further tested for promoting growth of oilseed rape in potting experiments. A total of 97 endophytic fungal isolates were obtained from roots (35), stems (49) and leaves (13) of B. napus. Forty fungal species were identified and most species (80%) belong to Ascomycota. The species composition is highly diversified with Simpson’s diversity index reaching 0.959. Alternaria alternata is the dominant species accounting for 12.4% of the isolates. Twenty-four isolates exhibited antifungal activity against S. sclerotiorum in dual cultures on potato dextrose agar forming inhibition zones of 3–17 mm in width. The culture filtrates of Aspergillus flavipes CanS-34A, Chaetomium globosum CanS-73, Clonostachys rosea CanS-43 and Leptosphaeria biglobosa CanS-51 in potato dextrose broth exhibited consistent and effective suppression of oilseed rape leaf blight caused by S. sclerotiorum. Fusarium oxysporum CanR-46 was detected capable of production of volatile organic compounds highly inhibitory to S. sclerotiorum and B. cinerea. Moreover, A. alternata CanL-18, Fusarium tricinctum CanR-70 and CanR-71r, and L. biglobosa CanS-51 exhibited growth-promoting effects on oilseed rape. These results suggest that B. napus harbors diversified endophytic fungi, from which potential biocontrol agents against S. sclerotiorum and B. cinerea, and for promoting growth of B. napus can be screened.     

Biological control of postharvest diseases of apples,peaches and nectarines by Bacillus subtilis S16 isolated from halophytes rhizosphere

Penicillium expansum, Botrytis cinerea and Colletotrichum acutatum are the most common postharvest pathogens of apples, peaches and nectarines. In this study, 96 bacteria were isolated from halophytes rhizosphere and assayed for biocontrol activity under in vitro conditions. Among the 96 isolates tested, isolate S16 effectively inhibited the growth of P. expansum, B. cinerea and C. acutatum. The isolate S16 has reduced 78.33±1.53 to 82.98±2.13% of disease severity in apples, peaches and nectarines. Matrix-assisted laser desorption ionisation-time of flight mass spectrometry of the antifungal compounds revealed three lipopeptide complexes, namely surfactins, iturins and fengycins. Lipopeptides and hydrolytic enzymes produced by the isolate S16 play an important role in the antifungal activity. Polymerase chain reaction analysis using ituD, srfAD, fenD and fenE gene-specific primers showed that the isolate S16 carry sequences similar to ituD, srfAD, fenD and fenE genes. Based on the 16S rDNA sequencing, the effective isolate S16 was identified as Bacillus subtilis.     

Molecular and Functional Characterization of a Polygalacturonase-Inhibiting Protein from Cynanchum komarovii That Confers Fungal Resistance in Arabidopsis

Compliance with ethical standards: This study did not involve human participants and animals, and the plant of interest is not an endangered species.Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat proteins that plants produce against polygalacturonase, a key virulence agent in pathogens. In this paper, we cloned and purified CkPGIP1, a gene product from Cynanchum komarovii that effectively inhibits polygalacturonases from Botrytis cinerea and Rhizoctonia solani. We found the expression of CkPGIP1 to be induced in response to salicylic acid, wounding, and infection with B. cinerea and R. solani. In addition, transgenic overexpression in Arabidopsis enhanced resistance against B. cinerea. Furthermore, CkPGIP1 obtained from transgenic Arabidopsis inhibited the activity of B. cinerea and R. solani polygalacturonases by 62.7–66.4% and 56.5–60.2%, respectively. Docking studies indicated that the protein interacts strongly with the B1-sheet at the N-terminus of the B. cinerea polygalacturonase, and with the C-terminus of the polygalacturonase from R. solani. This study highlights the significance of CkPGIP1 in plant disease resistance, and its possible application to manage fungal pathogens.     

Synergistic Activity of the Plant Defensin HsAFP1 and Caspofungin against Candida albicans Biofilms and Planktonic Cultures

Plant defensins are small, cysteine-rich peptides with antifungal activity against a broad range of yeast and fungi. In this study we investigated the antibiofilm activity of a plant defensin from coral bells (Heuchera sanguinea), i.e. HsAFP1. To this end, HsAFP1 was heterologously produced using Pichia pastoris as a host. The recombinant peptide rHsAFP1 showed a similar antifungal activity against the plant pathogen Fusarium culmorum as native HsAFP1 purified from seeds. NMR analysis revealed that rHsAFP1 consists of an α-helix and a triple-stranded antiparallel β-sheet stabilised by four intramolecular disulfide bonds. We found that rHsAFP1 can inhibit growth of the human pathogen Candida albicans as well as prevent C. albicans biofilm formation with a BIC50 (i.e. the minimum rHsAFP1 concentration required to inhibit biofilm formation by 50% as compared to control treatment) of 11.00 ± 1.70 μM. As such, this is the first report of a plant defensin exhibiting inhibitory activity against fungal biofilms. We further analysed the potential of rHsAFP1 to increase the activity of the conventional antimycotics caspofungin and amphotericin B towards C. albicans. Synergistic effects were observed between rHsAFP1 and these compounds against both planktonic C. albicans cells and biofilms. Most notably, concentrations of rHsAFP1 as low as 0.53 μM resulted in a synergistic activity with caspofungin against pre-grown C. albicans biofilms. rHsAFP1 was found non-toxic towards human HepG2 cells up to 40 μM, thereby supporting the lack of a general cytotoxic activity as previously reported for HsAFP1. A structure-function study with 24-mer synthetic peptides spanning the entire HsAFP1 sequence revealed the importance of the γ-core and its adjacent regions for HsAFP1 antibiofilm activity. These findings point towards broad applications of rHsAFP1 and its derivatives in the field of antifungal and antibiofilm drug development.     

Coordinate Accumulation of Antifungal Proteins and Hexoses Constitutes a Developmentally Controlled Defense Response during Fruit Ripening in Grape

During ripening of grape (Vitis labruscana L. cv Concord) berries, abundance of several proteins increased, coordinately with hexoses, to the extent that these became the predominant proteins in the ovary. These proteins have been identified by N-terminal amino acid-sequence analysis and/or function to be a thaumatin-like protein (grape osmotin), a lipid-transfer protein, and a basic and an acidic chitinase. The basic chitinase and grape osmotin exhibited activities against the principal grape fungal pathogens Guignardia bidwellii and Botrytis cinerea based on in vitro growth assays. The growth-inhibiting activity of the antifungal proteins was substantial at levels comparable to those that accumulate in the ripening fruit, and these activities were enhanced by as much as 70% in the presence of 1 m glucose, a physiological hexose concentration in berries. The simultaneous accumulation of the antifungal proteins and sugars during berry ripening was correlated with the characteristic development of pathogen resistance that occurs in fruits during ripening. Taken together, accumulation of these proteins, in combination with sugars, appears to constitute a novel, developmentally regulated defense mechanism against phytopathogens in the maturing fruit.Plants have evolved a number of strategies to resist fungal infection. One strategy involves the accumulation of defense proteins that have direct inhibitory activity against the hyphae and/or germinating spores of the pathogen. Among these are PR proteins including chitinases (PR-3 family), thaumatin-like proteins (PR-5 family), and nsLTPs. Typically, these antifungal proteins are expressed constitutively at low levels in cells and accumulate in response to fungal attack or in response to other inducers of acquired resistance (Uknes et al., 1992). Reproductive organs are apparently an exception to induced acquired resistance. Presumably, the importance of flowers and ovaries to the maintenance of the species has mandated that reproductive organs acquire pathogen resistance during development. Developmentally regulated expression of PR proteins has been observed in floral organs (Lotan et al., 1989; Neal et al., 1990), including numerous examples of defensive gene mRNA accumulation in fruits. However, comparatively few data are available that interrelate developmental accumulation of antifungal proteins and the acquisition of resistance against fruit pathogens (Fils-Lycaon et al., 1996; Meyer et al., 1996).Another physiological adaptation of plants that affects fungal pathogenesis, but one that has received considerably less attention, is the accumulation of sugars. Results from studies of several host/pathogen systems have implicated accumulation or depletion of sugars in resistance to fungal infection (VanderPlank, 1984). Increased susceptibility to fungi in sugar-depleted vegetative tissues, a phenomenon termed “sink-induced loss of resistance,” has been documented in tomato (Horsfall, 1975), cotton (Eaton and Rigler, 1946), and maize (Holbert et al., 1935). Conversely, moderate levels of sugars can enhance colonization rates of some fungal pathogens, presumably because these are important sources of C for the microbes (Mains, 1917). However, higher levels of sugars can reverse this effect, leading to decreased susceptibility, a phenomenon termed “high-sugar resistance” (Horsfall and Dimond, 1957). This reversal of susceptibility has been suggested to occur as a result of high sugar levels furnishing an osmotic challenge to the fungi and suppressing their colonization of the plant (VanderPlank, 1984).We report here that accumulation of hexoses in grape (Vitis labruscana L. cv Concord) berries is accompanied by a developmental-stage-specific increase in a suite of proteins that are homologous to proteins known to be antifungal determinants. These proteins have been identified as a thaumatin-like protein (Salzman et al., 1994) (here we have named it GO), chitinases (both CBC and AC forms), and a nsLTP. Physiological levels of GO or CBC assayed in vitro alone or in combination with physiological levels of Glc exhibited individual and/or combinatorial activities against the important grape pathogens Guignardia bidwellii, the causal agent of black rot, and Botrytis cinerea. The interaction of the antifungal proteins and hexoses appears to constitute a developmentally regulated defense mechanism to restrict fungal pathogen infection as seeds are maturing in ripening berries.