Nep1-like proteins as a target for plant pathogen control.

The lack of efficient methods to control the major diseases of crops most important to agriculture leads to huge economic losses and seriously threatens global food security. Many of the most important microbial plant pathogens, including bacteria, fungi, and oomycetes, secrete necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs), which critically contribute to the virulence and spread of the disease. NLPs are cytotoxic to eudicot plants, as they disturb the plant plasma membrane by binding to specific plant membrane sphingolipid receptors. Their pivotal role in plant infection and broad taxonomic distribution makes NLPs a promising target for the development of novel phytopharmaceutical compounds. To identify compounds that bind to NLPs from the oomycetes Pythium aphanidermatum and Phytophthora parasitica, a library of 587 small molecules, most of which are commercially unavailable, was screened by surface plasmon resonance. Importantly, compounds that exhibited the highest affinity to NLPs were also found to inhibit NLP-mediated necrosis in tobacco leaves and Phytophthora infestans growth on potato leaves. Saturation transfer difference-nuclear magnetic resonance and molecular modelling of the most promising compound, anthranilic acid derivative, confirmed stable binding to the NLP protein, which resulted in decreased necrotic activity and reduced ion leakage from tobacco leaves. We, therefore, confirmed that NLPs are an appealing target for the development of novel phytopharmaceutical agents and strategies, which aim to directly interfere with the function of these major microbial virulence factors. The compounds identified in this study represent lead structures for further optimization and antimicrobial product development.

Enhancing the Potentiality of Trichoderma harzianum against Pythium Pathogen of Beans Using Chamomile (Matricaria chamomilla, L.) Flower Extract.

Our present study was designed to investigate the role of both Trichoderma harzianum and chamomile (Matricaria chamomilla L.) flower extract in mutual reaction against growth of Pythium ultimum. In vitro, the activity of chamomile extract was found to reduce the radial growth of Pythium ultimum up to 30% compared to the control. Whereas, the radial growth reduction effect of T. harzianum against P. ultimum reached 81.6% after 120 h. Data also showed the productivity of total phenolics and total flavonoids by T. harzianum, was 12.18 and 6.33 mg QE/100 mL culture filtrate, respectively. However, these compounds were determined in chamomile flower extract at concentrations of 75.33 and 24.29 mg QE/100 mL, respectively. The fractionation of aqueous extract of chamomile flower using HPLC provided several polyphenolic compounds such as pyrogallol, myricetin, rosemarinic acid, catechol, p-coumaric acid, benzoic acid, chlorogenic acid and other minor compounds. In vivo, the potentiality of T. harzianum with chamomile flower extract against Pythium pathogen of bean was investigated. Data obtained showed a reduction in the percentage of rotted seed and infected seedling up to 28 and 8%, respectively. Whereas, the survival increased up to 64% compared to other ones. There was also a significant promotion in growth features, total chlorophyll, carotenoids, total polyphenols and flavonoids, polyphenol-oxidase and peroxidase enzymes compared to other ones. To the best of our knowledge, there are no reported studies that included the mutual association of fungus, T. harzianum with the extract taken from the chamomile flower against P. ultimum, either in vitro or in vivo. In conclusion, the application of both T. harzianum and/or M. chamomilla extracts in the control of bean Pythium pathogen showed significant results.

Feasibility of Volatile Biomarker-Based Detection of Pythium Leak in Postharvest Stored Potato Tubers Using Field Asymmetric Ion Mobility Spectrometry.

The study evaluates the suitability of a field asymmetric ion mobility spectrometry (FAIMS) system for early detection of the Pythium leak disease in potato tubers simulating bulk storage conditions. Tubers of Ranger Russet (RR) and Russet Burbank (RB) cultivars were inoculated with Pythium ultimum, the causal agent of Pythium leak (with negative control samples as well) and placed in glass jars. The headspace in sampling jars was scanned using the FAIMS system at regular intervals (in days up to 14 and 31 days for the tubers stored at 25 °C and 4 °C, respectively) to acquire ion mobility current profiles representing the volatile organic compounds (VOCs). Principal component analysis plots revealed that VOCs ion peak profiles specific to Pythium ultimum were detected for the cultivars as early as one day after inoculation (DAI) at room temperature storage condition, while delayed detection was observed for tubers stored at 4 °C (RR: 5th DAI and RB: 10th DAI), possibly due to a slower disease progression at a lower temperature. There was also some overlap between control and inoculated samples at a lower temperature, which could be because of the limited volatile release. Additionally, data suggested that the RB cultivar might be less susceptible to Pythium ultimum under reduced temperature storage conditions. Disease symptom-specific critical compensation voltage (CV) and dispersion field (DF) from FAIMS responses were in the ranges of -0.58 to -2.97 V and 30-84% for the tubers stored at room temperature, and -0.31 to -2.97 V and 28-90% for reduced temperature, respectively. The ion current intensities at -1.31 V CV and 74% DF showed distinctive temporal progression associated with healthy control and infected tuber samples.

Efficacy of miltefosine therapy against subcutaneous experimental pythiosis in rabbits.

We evaluated the in vitro activity of miltefosine against 29 Pythium spp. and the in vivo therapeutic response of 2mg/kg/day of miltefosine given orally to rabbit with pythiosis induced experimentally. The MICs (in µg/mL) of miltefosine was medium-dependent and ranged from 0.5 to 2 and 32-64 on RPMI 1640 and Mueller Hinton broth, respectively. The treatment with miltefosine demonstrated significantly lower subcutaneous lesion areas compared to the control group but was not sufficient for the complete remission of the lesions. This study indicates that miltefosine has limited efficacy against pythiosis and furthers in vitro and in vivo studies are necessary to determine the possible potential of this drug in the treatment of pythiosis.

Oils extracted from Eupatorium adenophorum leaves show potential to control Phythium myriotylum in commercially-grown ginger.

Oils extracted from the leaves of Eupatorium adenophorum were tested in vitro and in vivo against the soilborne pathogen Pythium myriotylum which causes soft rot, a devastating disease of commercial ginger production in China. Twelve compounds accounting for 99.15% of the total oil composition were identified by GC-MS. The major components were 10Hß-9-oxo-agerophorone (37.03%), 10Hα-9-oxo-agerophorone (37.73%) and 9-oxo-10, 11-dehydro-agerophorone (23.41%). Antifungal activity was tested by the poisoned food technique against P. myriotylum, indicating minimum inhibitory concentrations of 100µg/ml after 7 days incubation. In addition, the oil extracts greatly inhibited the formation of both wet and dry mycelial biomass. The combination of E. adenophorum oil extracts and synthetic fungicides showed a strong synergistic effect, inhibiting the mycelial growth in in vitro assays. The synergistic effect of oil extracts with fungicides could allow fungicides to be used at reduced rates in the future which has environmental advantages. Oil extracts applied at 160 and 200µg/ml concentrations to ginger rhizomes before inoculation with P. myriotylum significantly reduced the infection rate in ginger. Examination by light and transmission electron microscopy revealed that oil extracts caused swelling of the hyphae, disruption of the cell wall, degradation of the cytoplasmic organelles and shortening of the cytoplasmic inclusion. These results suggested that the plasma membrane and endomembrane systems of P. myriotylum were severely damaged by the oil extracts of E. adenophorum which offer significant potential for use as a fungicide to control P. myriotylum.

The use of newly isolated Streptomyces strain TN258 as potential biocontrol agent of potato tubers leak caused by Pythium ultimum.

An actinomycete strain designated TN258, was isolated from Tunisian Sahara soil and selected for its antagonistic activity, especially against Pythium ultimum (P. ultimum) causing potato tubers leak. Based on the results of cultural characteristic of TN258 strain, the 16S rRNA gene nucleotide sequence (1433 bp, accession n° HE600071) and the phylogenetic analysis, we propose the assignment of our new isolate bacterium as Streptomyces TN258 strain. After culture optimization, the inhibitory effect of TN258 free cell supernatant against P. ultimum was evaluated. As result, by application of 50% (v/v) from 25 mg ml-1 of concentration, mycelial growth was totally inhibited with hyphal destruction. At the same concentration, the oospores were distorted and the germination was completely stopped. In potato tubers, Streptomyces TN258 filtrated supernatant, applied 24 h before inoculation by P. ultimum (preventive treatment group) was able to significantly decrease pathogen penetration by 62% and to reduce the percentage of weight loss by 59.43%, in comparison with non-treated group.

Chlorophyll fluorescence imaging as a tool to monitor the progress of a root pathogen in a perennial plant.

MAIN CONCLUSION: The chlorophyll fluorescence parameter ΦNO is an excellent metric for the non-destructive monitoring of disease progression, measured over a broad range of light intensities. The suitability of the slow induction chlorophyll fluorescence parameters ΦPSII, ΦNPQ, and ΦNO to monitor in vivo disease progression in a host-root pathogen pathosystem was evaluated and compared to the established method of monitoring disease by measuring Fv/Fm. Using the infection of ginseng plants (Panax quinquefolius L.) with Pythium irregulare Buisman as a model, light response curves were used to establish the optimal irradiance for the resolution of differences between fluorescence parameters ΦPSII, ΦNPQ and ΦNO. As infection progressed only changes in ΦNO remained consistent with increased irradiance, and increased as infection progressed. Furthermore, ΦNO showed a high sensitivity for distinguishing increased disease load. In contrast, the magnitude in change of ΦPSII and ΦNPQ were sensitive to irradiance levels. The magnitude of increase in ΦNO per unit disease score was equivalent to the corresponding decline in Fv/Fm values. Thus ΦNO is as sensitive as Fv/Fm in monitoring biotic stress. The ability to measure ΦNO under a wide range of light intensities, including natural light, potentially without the need for dark adaptation, means that it can be used in the development of a general protocol for non-invasive, in vivo monitoring of plant health, from the laboratory to the field scale.

The biosurfactant viscosin produced by Pseudomonas fluorescens SBW25 aids spreading motility and plant growth promotion.

Food security depends on enhancing production and reducing loss to pests and pathogens. A promising alternative to agrochemicals is the use of plant growth-promoting rhizobacteria (PGPR), which are commonly associated with many, if not all, plant species. However, exploiting the benefits of PGPRs requires knowledge of bacterial function and an in-depth understanding of plant-bacteria associations. Motility is important for colonization efficiency and microbial fitness in the plant environment, but the mechanisms employed by bacteria on and around plants are not well understood. We describe and investigate an atypical mode of motility in Pseudomonas fluorescens SBW25 that was revealed only after flagellum production was eliminated by deletion of the master regulator fleQ. Our results suggest that this 'spidery spreading' is a type of surface motility. Transposon mutagenesis of SBW25ΔfleQ (SBW25Q) produced mutants, defective in viscosin production, and surface spreading was also abolished. Genetic analysis indicated growth-dependency, production of viscosin, and several potential regulatory and secretory systems involved in the spidery spreading phenotype. Moreover, viscosin both increases efficiency of surface spreading over the plant root and protects germinating seedlings in soil infected with the plant pathogen Pythium. Thus, viscosin could be a useful target for biotechnological development of plant growth promotion agents.

Influence of phosphorus and pH on the fungicidal potential of Anabaena strains.

The genus Anabaena is known to be a rich source of bioactive metabolites, but the biocontrol potential of this genus, mediated through hydrolytic enzymes is less investigated. In our investigation, five Anabaena strains - A. laxa RPAN8, A. iyengarii RPAN9, A. variabilis RPAN59 and A. oscillarioides RPAN69 (with A. variabilis RPAN16 serving as negative control) were evaluated in time course studies involving incubation under three levels of phosphorus and pH conditions. Total chlorophyll, proteins, chitosanase, endoglucanase and CMCase activity were measured and inhibition assayed against phytopathogenic fungi. The four weeks old RPAN69 culture showed significantly higher chlorophyll which was 41% higher than control. This was also linked with an enhancement of 18.26% and 9.18% in chitosanase and CMCase activity respectively over control in the treatment involving half dose of phosphorus. Chlorophyll and CMCase activity showed a high degree of correlation with highest values at pH 9.5. A pH of 5.5 was the most suitable condition for the maximum activity of chitosanase for all the strains except RPAN16. The strains RPAN8 and RPAN9 showed the highest activity of endoglucanase at pH 5.5 while the other strains exhibited maximum activity at pH 7.5. This study provides insight into the role of P and pH in modulating fungicidal activity in different Anabaena strains, which can be valuable for enhancing their efficiency as a biocontrol agent.

Ginsenosidases and the pathogenicity of Pythium irregulare.

American ginseng (Panax quinquefolius L.) produces triterpenoid saponins, ginsenosides, that possess mild fungitoxic activity toward some common ginseng leaf pathogens. However, numerous oomycete root pathogens of ginseng, most notably Pythium irregulare Buisman, are able to partially deglycosylate 20 (S)-protopanaxadiol ginsenosides Rb1, Rd and gypenoside XVII via extracellular glycosidases, leading to a common product, ginsenoside F2. Conversion of the common 20 (S)-protopanaxadiols into F2 requires both ß (1→6) and ß (1→2) glucosidase activity. In the present study, the ability of nine distinct isolates of P. irregulare, as well as a P. ultimum Trow isolate and two isolates of Trichoderma hamatum (Bonord.) Bainier, to deglycosylate 20 (S)-protopanaxadiols, in vitro was examined. The pathogenicity of each isolate was also examined by scoring the severity of disease symptoms caused by each in separate inoculations of one- and two-year old ginseng seedlings. Disease severity was scored using a disease severity index, as well as by taking F(v)/F(m) measurements of leaves during a 14-day infection period. Based on these measurements, it was concluded that (1) the use of direct F(v)/F(m) measurements correlates strongly with observations of disease severity (R(2)=0.79), and that (2) the pathogenicity of P. irregulare isolates correlates with their ability to deglycosylate ginsenosides (R(2)=0.57). These results further support the hypothesis that the pathogenicity of P. irregulare on ginseng roots is dependent, in part, on the ability of this organism to deglycosylate ginsenosides.

Microbial-induced carbon competition in the spermosphere leads to pathogen and disease suppression in a municipal biosolids compost.

The aim of this study was to understand whether competition for fatty acids in plant seed exudates by compost-derived seed-colonizing microbial communities could explain the suppression of plant infections initiated by sporangia of Pythium ultimum. The germination behavior of P. ultimum sporangia in response to cucumber seeds was measured to determine the impact of seed-colonizing microbes on pathogen suppression. Seed-colonizing microbial communities from municipal biosolids compost utilized cucumber seed exudates and linoleic acid in vitro, reducing the respective stimulatory activity of these elicitors to P. ultimum sporangial germination. However, when sporangia were observed directly in the spermosphere of seeds sown in the compost medium, levels of germination and sporangial emptying did not differ from the responses in sand. The percentage of aborted germ tubes was greater after incubating sporangia in compost medium for 12-h than the level of germ tube abortion when sporangia were incubated in sand. Abortion did not occur if previously germinated sporangia were supplemented with cucumber seed exudate. Furthermore, removal of cucumber seed exudate after various stages of germ tube emergence resulted in an increase in aborted germ tubes over time. Adding increasing levels of glucose directly to the compost medium alleviated germ tube abortion in the spermosphere and also eliminated disease suppression. These data fail to support a role for linoleic acid competition in Pythium seedling disease suppression but provide evidence for general carbon competition mediated by seed-colonizing microbial communities as a mechanism for the suppression of Pythium seed infections in municipal biosolids compost.

Mutation of a degS homologue in Enterobacter cloacae decreases colonization and biological control of damping-off on cucumber.

We have been using mutagenesis to determine how biocontrol bacteria such as Enterobacter cloacae 501R3 deal with complex nutritional environments found in association with plants. E. cloacae C10, a mutant of 501R3 with a transposon insertion in degS, was diminished in growth on synthetic cucumber root exudate (SRE), colonization of cucumber seed and roots, and control of damping-off of cucumber caused by Pythium ultimum. DegS, a periplasmic serine protease in the closely related bacterium Escherichia coli K12, is required for the RpoE-mediated stress response. C10 containing wild-type degS from 501R3 or from E. coli K12 on pBeloBAC11 was significantly increased in growth on SRE, colonization of cucumber roots, and control of P. ultimum relative to C10 containing pBeloBAC11 alone. C10 and 501R3 were similar in sensitivity to acidic conditions, plant-derived phenolic compounds, oxidative stress caused by hydrogen peroxide, dessication, and high osmoticum; stress conditions potentially associated with plants. This study demonstrates a role for degS in the spermosphere and rhizosphere during colonization and disease control by Enterobacter cloacae. This study implicates, for the first time, the involvement of DegS and, by extension, the RpoE-mediated stress response, in reducing stress on E. cloacae resulting from the complex nutritional environments in the spermosphere and rhizosphere.

Sugar beet-associated bacterial and fungal communities show a high indigenous antagonistic potential against plant pathogens.

The aim of this study was to analyze microbial communities in/on sugar beet with special focus on antagonists toward plant pathogens. For this purpose, the composition of microorganisms isolated from the rhizosphere, phyllosphere, endorhiza, and endosphere of field-grown sugar beet plants was analyzed by a multiphasic approach at three different plant development stages at six locations in Europe. The analysis of microbial communities by Single Strand Conformation Polymorphism (SSCP) of 16S/18S rRNA clearly revealed the existence of discrete microenvironment- and site-specific patterns. A total of 1952 bacterial and 1344 fungal isolates screened by dual testing for antagonism toward the pathogens Aphanomyces cochlioides, Phoma betae, Pythium ultimum, and Rhizoctonia solani resulted in 885 bacterial (=45%) and 437 fungal (=33%) antagonists. In general, the indigenous antagonistic potential was very high and influenced by (a) the location, (b) the plant developmental stage, and (3) the microenvironment. Furthermore, we showed for the first time that the antagonistic potential was highly specific for each target pathogen. The majority of antagonistic microorganisms suppressed only one pathogen (bacteria: 664 = 75%; fungi: 256 = 59%), whereas the minority showed a broad host range (bacteria: 4 = 0.5%; fungi: 7 = 1.6%). The bacterial communities harbored the highest antagonistic potential against P. ultimum, whereas the fungal communities contained more antagonists against A. cochlioides and R. solani. In contrast to their high proportion, only a low diversity of antagonists at genotypic and species level was found. Novel antagonistic species, e.g., Subtercola pratensis or Microbacterium testaceum were found in the internal part of the sugar beet body.

Loss of pectin is an early event during infection of cocoyam roots by Pythium myriotylum.

Cocoyam (Xanthosoma sagittifolium) is an important tuber crop in most tropical zones of Africa and America. In Cameroon, its cultivation is hampered by a soil-borne fungus Pythium myriotylum which is responsible for root rot disease. The mechanism of root colonisation by the fungus has yet to be elucidated. In this study, using microscopical and immunocytochemical methods, we provide a new evidence regarding the mode of action of the fungus and we describe the reaction of the plant to the early stages of fungal invasion. We show that the fungal attack begins with the colonisation of the peripheral and epidermal cells of the root apex. These cells are rapidly lost upon infection, while cortical and stele cells are not. Labelling with the cationic gold, which binds to negatively charged wall polymers such as pectins, is absent in cortical cells and in the interfacial zone of the infected roots while it is abundant in the cell walls of stele cells. A similar pattern of labelling is also found when using the anti-pectin monoclonal antibody JIM5, but not with anti-xyloglucan antibodies. This suggests that early during infection, the fungus causes a significant loss of pectin probably via degradation by hydrolytic enzymes that diffuse and act away from the site of attack. Additional support for pectin loss is the demonstration, via sugar analysis, that a significant decrease in galacturonic acid content occurred in infected root cell walls. In addition, we demonstrate that one of the early reactions of X. sagittifolium to the fungal invasion is the formation of wall appositions that are rich in callose and cellulose.

Zoospore encystment and pathogenicity of Phytophthora and Pythium species on plant roots.

Seven plant species (lucerne, maize, oat, sugarbeet, sorghum, tomato, wheat) and 12 Pythium and Phytophthora species were used in a comparative study designed to investigate the effects of plant and oomycete inter-specific variation on zoospore encystment density and pathogenicity. Zoospores showed differential encystment behaviour and they encysted more on dicotyledonous than on monocotyledonous plants. Pythium aphanidermatum, P. deliense, and Phytophthora nicotianae were the most aggressive species. Sugarbeet was the most severely attacked plant species followed by tomato while oat plants were relatively unaffected. The relationship between zoospore encystment on roots and disease severity depended on the oomycete-plant combination. Correlation analysis between zoospore encystment density and disease severity indicated low and no significant levels (p.05) of association for most plant-oomycete combinations.

A clp gene homologue belonging to the Crp gene family globally regulates lytic enzyme production, antimicrobial activity, and biological control activity expressed by Lysobacter enzymogenes strain C3.

Lysobacter enzymogenes strain C3, a biological control agent for plant diseases, produces multiple extracellular hydrolytic enzymes and displays antimicrobial activity against various fungal and oomycetous species. However, little is known about the regulation of these enzymes or their roles in antimicrobial activity and biocontrol. A study was undertaken to identify mutants of strain C3 affected in extracellular enzyme production and to evaluate their biocontrol efficacy. A single mini-Tn5-lacZ(1)-cat transposon mutant of L. enzymogenes strain C3 that was globally affected in a variety of phenotypes was isolated. In this mutant, 5E4, the activities of several extracellular lytic enzymes, gliding motility, and in vitro antimicrobial activity were reduced. Characterization of 5E4 indicated that the transposon inserted in a clp gene homologue belonging to the Crp gene family of regulators. Immediately downstream was a second open reading frame similar to that encoding acetyltransferases belonging to the Gcn5-related N-acetyltransferase superfamily, which reverse transcription-PCR confirmed was cotranscribed with clp. Chromosomal deletion mutants with mutations in clp and between clp and the acetyltransferase gene verified the 5E4 mutant phenotype. The clp gene was chromosomally inserted in mutant 5E4, resulting in complemented strain P1. All mutant phenotypes were restored in P1, although the gliding motility was observed to be excessive compared with that of the wild-type strain. clp mutant strains were significantly affected in biological control of pythium damping-off of sugar beet and bipolaris leaf spot of tall fescue, which was partially or fully restored in the complemented strain P1. These results indicate that clp is a global regulatory gene that controls biocontrol traits expressed by L. enzymogenes C3.

Inability to find consistent bacterial biocontrol agents of Pythium aphanidermatum in cucumber using screens based on ecophysiological traits.

A collection of 821 rhizobacteria from cucumber, originating from different root locations and stages of plant development, was screened for potential biocontrol agents of Pythium aphanidermatum (Edson) Fitzp. The screening procedure exploited carbon source utilization profiles and growth rates of bacteria as indicators of a partial niche overlap with the pathogen. The bacteria were tested for growth on nine carbon sources (glucose, fucose, sucrose, maltose, asparagine, alanine, galacturonic acid, succinic acid, and linoleic acid), most of which are reported to be used by the zoospores of P. aphanidermatum in the infection process. The isolates were classified as fast- or slow-growing, depending on their growth rate in 1/10 strength TSB. By nonhierarchical cluster analysis, 20 clusters were generated of bacteria with similar profiles of carbon source utilization. Redundancy analysis showed that the type of root sample explained 47% of the variance found in the relative abundance of bacteria from the clusters. Bacteria from clusters using none or few of the carbon sources, e.g., maltose and linoleic acid, with many slow-growing isolates, showed a preference for plants in the vegetative or generative stage, or for old root regions (root base). Bacteria from clusters with fast-growing isolates, using many carbon sources, were relatively abundant in the seedling stage. A selection of 127 bacteria from the different clusters was tested for disease suppressive capabilities in bioassays on young cucumber plants in nutrient solution, inoculated with zoospores of P. aphanidermatum. Nine of these bacteria produced biosurfactants, and 27 showed antibiosis against mycelial growth in plate assays. For 31 isolates, significant positive effects on plant biomass were shown, as analyzed with a general linear regression model. For most isolates, these effects occurred only in one of two replicate assays and no reductions in the degree of root and crown rot were found. Of the isolates that used many of the tested carbon sources, only four had positive effects on plant biomass. The majority of the isolates that positively affected plant biomass used few to moderate numbers of carbon sources and did not produce antibiotics or biosurfactants. In conclusion, competition for the tested carbon sources with the zoospores did not play a decisive role in disease suppression, and no clear relation was found between ecophysiological traits and disease suppression. Only isolate 3.1T8, isolated from root tips in the generative stage of plant growth, significantly increased plant biomass and suppressed root and crown rot symptoms in five out of six bioassays. The isolate produced an antifungal substance in plate assays and showed biosurfactant production in several (cucumber-derived) media.

Synthetic peptide combinatorial libraries: a method for the identification of bioactive peptides against phytopathogenic fungi.

Synthetic combinatorial libraries were evaluated with an iterative process to identify a hexapeptide with broadspectrum activity against selected phytopathogenic fungi. A D-amino acid hexapeptide (FRLKFH) and pentapeptide (FRLHF) exhibited activity against Fusarium oxysporum f. sp. lycopersici, Rhizoctonia solani (anastomosis group 1), Ceratocystis fagacearum, and Pythium ultimum. The peptides showed no hemolytic or mutagenic activity. Fluorescent microscopy studies with a membrane impermeant dye indicated that fungal cytoplasmic membranes were compromised rapidly and that the nuclear membrane was also affected.