Camalexin induces detoxification of the phytoalexin brassinin in the plant pathogen Leptosphaeria maculans

The impact of the phytoalexins camalexin and spirobrassinin on brassinin detoxification by Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.], a pathogenic fungus prevalent on crucifers, was investigated. Brassinin is a plant metabolite of great significance due to its dual role both as an effective phytoalexin and as an early biosynthetic precursor of the majority of the phytoalexins produced by plants of the family Brassicaceae (Cruciferae). The rate of detoxification of brassinin in cultures of L. maculans increased substantially in the presence of camalexin, whereas spirobrassinin did not appear to have a detectable effect. In addition, the brassinin detoxifying activity of cell-free extracts obtained from cultures incubated with camalexin was substantially higher than that of control cell-free extracts or cultures incubated with spirobrassinin, and correlated positively with brassinin oxidase activity. The discovery of a potent synthetic modulator of brassinin oxidase activity, 3-phenylindole, and comparison with the commercial fungicide thiabendazole is also reported. The overall results indicate that brassinin oxidase production is induced by camalexin and 3-phenylindole but not by spirobrassinin or thiabendazole. Importantly, our work suggests that introduction of the camalexin pathway into plants that produce brassinin might make these plants more susceptible to L. maculans.     

Polyphenoloxidase activity in coffee leaves and its role in resistance against the coffee leaf miner and coffee leaf rust

In plants, PPO has been related to defense mechanism against pathogens and insects and this role was investigated in coffee trees regarding resistance against a leaf miner and coffee leaf rust disease. PPO activity was evaluated in different genotypes and in relation to methyl-jasmonate (Meja) treatment and mechanical damage. Evaluations were also performed using compatible and incompatible interactions of coffee with the fungus Hemileia vastatrix (causal agent of the leaf orange rust disease) and the insect Leucoptera coffeella (coffee leaf miner). The constitutive level of PPO activity observed for the 15 genotypes ranged from 3.8 to 88 units of activity/mg protein. However, no direct relationship was found with resistance of coffee to the fungus or insect. Chlorogenic acid (5-caffeoylquinic acid), the best substrate for coffee leaf PPO, was not related to resistance, suggesting that oxidation of other phenolics by PPO might play a role, as indicated by HPLC profiles. Mechanical damage, Meja treatment, H. vastatrix fungus inoculation and L. coffeella infestation caused different responses in PPO activity. These results suggest that coffee resistance may be related to the oxidative potential of the tissue regarding the phenolic composition rather than simply to a higher PPO activity.     

Galiellalactone and its biogenetic precursors as chemotaxonomic markers of the Sarcosomataceae (Ascomycota)

(-)-Galiellalactone is a hexaketide metabolite with interesting pharmacological activities which was detected in four strains of Galiella rufa (Sarcosomataceae, Ascomycota) and in two unidentified fungi shown by their 18S rDNA sequences also to belong to the Sarcosomataceae. These were a wood-inhabiting apothecial species from Chile and an endophytic isolate from Cistus salviifolius (Sardinia). Other members of the family (Urnula helvelloides, one Strumella coryneoidea isolate) produced no galiellalactone but merely hexaketides structurally related to galiellalactone precursors, whereas a third group of species (Sarcosoma latahensis, Strumella griseola, one S. coryneoidea isolate) lacked hexaketide production altogether. Despite thorough screening programmes, galiellalactone and its precursors have not yet been found in any fungus outside the Sarcosomataceae and may thus be a chemotaxonomic marker of the family, supporting its current phylogenetic definition. Two pentaketide derivatives of the 6-pentyl-alpha-pyrone type were found in all G. rufa strains as well as in A111-95 and the hexaketide-producing S. coryneoidea isolate.     

The phytopathogenic fungus Alternaria brassicicola: Phytotoxin production and phytoalexin elicitation

The metabolites and phytotoxins produced by the phytopathogenic fungus Alternaria brassicicola (Schwein.) Wiltshire, as well as the phytoalexins induced in host plants, were investigated. Brassicicolin A emerged as the most selective phytotoxic metabolite produced in liquid cultures of A. brassicicola and spirobrassinin as the major phytoalexin produced in infected leaves of Brassica juncea (whole plants). In detached infected leaves of B. juncea, the main component was N′-acetyl-3-indolylmethanamine, the product of detoxification of the phytoalexin brassinin by A. brassicicola. In addition, the structure elucidation of three hitherto unknown metabolites having a fusicoccane skeleton was carried out and the antifungal activity of several plant defenses against A. brassicicola was determined.     

Drazepinone, a trisubstituted tetrahydronaphthofuroazepinone with herbicidal activity produced by Drechslera siccans

When grown in a minimal-defined medium, a strain of Drechslera siccans, a pathogenic fungus isolated from seeds of Lolium perenne, produced phytotoxic metabolites. This strain is one of the best toxin producers among several grass pathogenic fungal strains collected and tested to find phytotoxins to be used as natural herbicides of monocot weeds. From the culture filtrates of D. siccans, we isolated a new phytotoxic trisubstituted naphthofuroazepinone, named drazepinone, and characterised it as a 3,5,12a-trimethyl-2,5,5a,12a-tetrahydro-1H-naphtho[2',3':4,5]furo[2,3-b]azepin-2-one. Assayed at 2 microg microl(-1) solution the novel metabolite proved to have broad-spectrum herbicidal properties, without antibacterial and antifungal activities, and low zootoxic activity. Its original chemical structure and the interesting biological properties make drazepinone a potential natural herbicide.     

Acid phosphatase activity during the interaction of the nematophagous fungus Duddingtonia flagrans with the nematode Panagrellus sp

The use of Duddingtonia flagrans, a nematode-trapping fungus, has been investigated as a biological control method against free living larvae of gastrointestinal nematodes of livestock animals. This fungus captures and infects the nematode by cuticle penetration, immobilization and digestion of the internal contents. It has been suggested that this sequence of events occurs by a combination of physical and enzymatical activities. This report characterizes the acid phosphatase activity during the interaction of D. flagrans with the free-living nematode Panagrellus sp. The optimum pH for the hydrolysis of the acid phosphatase substrate p-nitrophenyl phosphate was 2.2, 2.8 and 5.4 from D. flagrans alone and 2.2 and 5.4 for Panagrellus sp alone, fungus-nematode interaction in liquid medium and fungus-nematode interaction in solid medium. Different acid phosphatase activity bands were detected by SDS-PAGE. Maximum acid phosphatase activity of the fungus or nematode alone and of the fungus-nematode interaction occurred within 70 min of incubation in the presence of the substrate 4-methylumbelliferyl phosphate. The activity of this enzyme was significantly higher for the fungus-nematode interaction when compared to the organisms alone, indicating a synergistic response. Furthermore, structures appeared in the hyphae after 30 min, nematodes were observed adhered after 40 min and many were captured by the typical fungus traps after 70 min of interaction. The participation of acid phosphatase activity and its importance during the interaction of the fungus with the nematode were discussed.     

The biotransformation of the diterpene 2beta-hydroxy-ent-13-epi-manoyl oxide by Gibberella fujikuroi

Incubation of the diterpene 2beta-hydroxy-ent-13-epi-manoyl oxide with Gibberella fujikuroi afforded in good yield 2beta,6beta-dihydroxy-ent-13-epi-manoyl oxide, 2beta,12beta-dihydroxy-ent-13-epi-manoyl oxide and 2beta,20-dihydroxy-ent-13-epi-manoyl oxide, confirming that although ent-13-epi-manoyl oxide is a final metabolite of a biosynthetic branch in this fungus, more polar derivatives of this compound can be transformed by this micro-organism.     

Chemical defenses of crucifers: elicitation and metabolism of phytoalexins and indole-3-acetonitrile in brown mustard and turnip

The metabolism of the cruciferous phytoalexins brassinin and cyclobrassinin, and the related compounds indole-3-carboxaldehyde, glucobrassicin, and indole-3-acetaldoxime was investigated in various plant tissues of Brassica juncea and B. rapa. Metabolic studies with brassinin showed that stems of B. juncea metabolized radiolabeled brassinin to indole-3-acetic acid, via indole-3-carboxaldehyde, a detoxification pathway similar to that followed by the "blackleg" fungus (Phoma lingam/Leptosphaeria maculans). In addition, it was established that tetradeuterated brassinin was incorporated into the phytoalexin brassilexin in B. juncea and B. rapa. On the other hand, the tetradeuterated indole glucosinolate glucobrassicin was not incorporated into brassinin, although the chemical structures of brassinins and indole glucosinolates suggest an interconnected biogenesis. Importantly, tetradeuterated indole-3-acetaldoxime was an efficient precursor of phytoalexins brassinin, brassilexin, and spirobrassinin. Elicitation experiments in tissues of Brassica juncea and B. rapa showed that indole-3-acetonitrile was an inducible metabolite produced in leaves and stems of B. juncea but not in B. rapa. Indole-3-acetonitrile displayed antifungal activity similar to that of brassilexin, was metabolized by the blackleg fungus at slower rates than brassinin, cyclobrassinin, or brassilexin, and appeared to be involved in defense responses of B. juncea.     

Effect of seasonal abiotic conditions and field margin habitat on the activity of Pandora neoaphidis inoculum on soil

The ability of the aphid pathogenic fungus Pandora neoaphidis to remain active in the absence of a resting stage through a combination of continuous infection and as conidia deposited on soil was assessed alongside the potential for planted field margins to act as a refuge for the fungus. P. neoaphidis was able to infect the pea aphid, Acyrthosiphon pisum, when maintained under controlled conditions that simulated those that occur seasonally in the UK. Although there was a significant inverse relationship between temperature and time-to-kill, with death occurring after 4.2, 6.9 and 13.6 days when maintained under fluctuating summer, autumn and winter temperatures, respectively, there were no additional statistically significant effects of photoperiod. The activity of inoculum on soil was indirectly assessed by baiting with A. pisum. Under controlled conditions P. neoaphidis remained active on soil and was able to infect aphids for up to 80 days. However, the percentage of aphids that became infected decreased from 76% on day 1 to 11% on day 80. Whereas there was little difference in the activity of conidia that had been maintained at 4 degrees C and 10 degrees C, activity at 18 degrees C was considerably reduced. Under field conditions the activity of inoculum was strongly influenced by season. On day 49 there was little or no activity during spring, summer or winter. However, during autumn a mean proportion of 0.08 aphids still became infected with P. neoaphidis. Margin type did not affect the activity of conidia nor was there a difference in activity between blocks that had regenerated naturally and those that had been planted. These results suggest that P. neoaphidis can infect aphids and remain active on soil under the abiotic conditions that occur seasonally in the UK and that this fungus may be able to persist annually without a resting stage.     

3-hydroxypropionic acid as a nematicidal principle in endophytic fungi

3- Hydroxypropionic acid was isolated by bioactivity-guided fractionation of extracts obtained from submerged cultures of several endophytic fungi isolated from above-ground plant organs. This compound showed selective nematicidal activity against the plant-parasitic nematode Meloidogyne incognita with LD50 values of 12.5-15 microg/ml. Activity against the saprophytic Caenorhabditis elegans was fivefold lower. No antimicrobial, cytotoxic or phytotoxic effects were observed. Propionic acid and D- and L-lactic acids were not active against either nematode species. Based on morphological features and ITS, 18S and 28S rDNA analyses, the producing strains were identified as Phomopsis phaseoli isolated from the leaf of a tropical tree, and four strains of Melanconium betulinum isolated from twigs of Betula pendula and B. pubescens in Germany. This is the first report of 3-hydroxypropionic acid in fungi, and of the nematicidal activity of this metabolite.     

Transmission of Pandora neoaphidis in the presence of co-occurring arthropods

Transmission of the entomopathogenic fungus Pandora neoaphidis to the nettle aphid Microlophium carnosum was assessed in the presence of arthropods that co-exist with the fungus within the habitat but do not compete for aphid hosts. The presence of a parasitoid significantly enhanced transmission, and transmission rates were similar for both enemy and non-enemy parasitoids. Although herbivory of nettle leaves by Peacock butterfly (Inchis io) caterpillars indirectly reduced the number of M. carnosum by >30% due to a reduction in leaf area for feeding, the addition of I. io significantly increased transmission of P. neoaphidis in the remaining aphids. It is likely that enhanced transmission in the presence of A. rhopalosiphii and I. io is due to disturbance and subsequent movement of the aphid, resulting in contact with conidia deposited on the leaf surface. The presence and impact of co-occurring arthropods should be taken into consideration when assessing the transmission of fungal entomopathogens.     

Characterization of the Biocidal Spectrum of Extracellular Filtrates of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis>

The effect of dichloromethane (DCM) and ethyl acetate (EA) extract of a cyanobacterium, Microcystis aeruginosa was evaluated against unicellular cyanobacteria and the phytopathogenic fungus Rhizoctonia solani. Fractionation of the filtrate showed the presence of five spots of different Rf values on silica gel coated plates indicating the presence of a number of compounds in the extract. A marked reduction in growth (52%) of the fungus was recorded on the plates supplemented with cyanobacterial extract, indicating the involvement of anti-fungal metabolite(s). The extract did not show any negative influence on seed germination and growth of seedlings of wheat, rice and mung, emphasizing the suitability of the compound for use in agriculture.     

Survival of Atta sexdens workers on different food sources

Leaf-cutting ants belonging to the tribe Attini are major herbivores and important agriculture pests in the neotropics, these ants being thought to feed on the sap which exudes from the plant material which they cut and also on the mycelium of a symbiotic fungus that grows on plant material inside their nests in what is called "the fungus garden". However, we have found that the survival of Atta sexdens worker ants on leaves, on mycelium of the ants' symbiotic fungus, Leucoagaricus gongylophorus, or on plant polysaccharides was the same as that of starved A. sexdens, while, conversely, significantly longer survival was achieved by ants fed on the fungus garden material or on some of the products (especially glucose) of the hydrolysis of plant polysaccharides. We found that the fungus garden contained glucose at a higher concentration than that found in leaves or fungal mycelium, and that this glucose was consumed by the ant to the extent that it was probably responsible for up to 50% of the nutritional needs of the workers. The fungus garden contained polysaccharide degrading enzymes (pectinase, amylase, xylanase and cellulase) in proportions similar to that observed in laboratory cultures of L. gongylophorus. It thus appears that A. sexdens workers obtain a significant part of their nutrients from plant polysaccharide hydrolysis products produced by the action of extracellular enzymes released by L. gongylophorus. In this paper we discuss the symbiotic nutrition strategy of A. sexdens workers and brood and the role played by plant polysaccharides in the nutrition of attine ants.     

Response of the entomopathogenic fungus Pandora neoaphidis to aphid-induced plant volatiles

We used a model plant-aphid system to investigate whether the aphid-specific entomopathogenic fungus Pandora neoaphidis responds to aphid-induced defence by the broad-bean plant, Vicia faba. Laboratory experiments indicated that neither in vivo sporulation, conidia size nor the in vitro growth of P. neoaphidis was affected by Acyrthosiphon pisum-induced V. faba volatiles. The proportion of conidia germinating on A. pisum feeding on previously damaged plants was significantly greater than on aphids feeding on undamaged plants, suggesting a direct functional effect of the plant volatiles on the fungus. However, there were no significant differences in the infectivity of P. neoaphidis towards A. pisum feeding on either undamaged V. faba plants or plants previously infested with A. pisum. Therefore, these results provide no evidence to suggest that P. neoaphidis contributes to plant indirect defence strategies.     

Phytochemistry and antimycobacterial activity of Chlorophytum inornatum

In a project to investigate plant derived natural products from the Liliaceae with activity against fast-growing strains of mycobacteria, we have identified two new metabolites from Chlorophytum inornatum. The active principle, a new homoisoflavanone (1) was identified as 3-(4'-methoxybenzyl)-7,8-methylenedioxy-chroman-4-one. The metabolite assigned as 7-(1'-hydroxyethyl)-2-(2'-hydroxyethyl)-3,4-dihydrobenzopyran (2) was characterised by extensive 1- and 2D NMR spectroscopy. The antimycobacterial activity of this plant was mainly due to the homoisoflavonoid which exhibited minimum inhibitory values ranging from 16-256 microg/ml against four strains of fast-growing mycobacteria.     

Vascular Streak Dieback of cacao in Southeast Asia and Melanesia: in planta detection of the pathogen and a new taxonomy

Vascular Streak Dieback (VSD) disease of cacao (Theobroma cacao) in Southeast Asia and Melanesia is caused by a basidiomycete (Ceratobasidiales) fungus Oncobasidium theobromae (syn. =Thanatephorus theobromae). The most characteristic symptoms of the disease are green-spotted leaf chlorosis or, commonly since about 2004, necrotic blotches, followed by senescence of leaves beginning on the second or third flush behind the shoot apex, and blackening of infected xylem in the vascular traces at the leaf scars resulting from the abscission of infected leaves. Eventually the shoot apex is killed and infected branches die. In susceptible cacao the fungus may grow through the xylem down into the main stem and kill a mature cacao tree. Infections in the stem of young plants prior to the formation of the first 3-4 lateral branches usually kill the plant. Basidiospores released from corticioid basidiomata developed on leaf scars or along cracks in the main vein of infected leaves infect young leaves. The pathogen commonly infects cacao but there are rare reports from avocado. As both crops are introduced to the region, the pathogen is suspected to occur asymptomatically in native vegetation. The pathogen is readily isolated but cultures cannot be maintained. In this study, DNA was extracted from pure cultures of O. theobromae obtained from infected cacao plants sampled from Indonesia. The internal transcribed spacer region (ITS), consisting of ITS1, 5.8S ribosomal RNA and ITS2, and a portion of nuclear large subunit (LSU) were sequenced. Phylogenetic analysis of ITS sequences placed O. theobromae sister to Ceratobasidium anastomosis groups AG-A, AG-Bo, and AG-K with high posterior probability. Therefore the new combination Ceratobasidium theobromae is proposed. A PCR-based protocol was developed to detect and identify C. theobromae in plant tissue of cacao enabling early detection of the pathogen in plants. A second species of Ceratobasidium, Ceratobasidium ramicola, identified through ITS sequence analysis, was isolated from VSD-affected cacao plants in Java, and is widespread in diseased cacao collected from Indonesia.     

Entomophthora leyteensis Villacarlos & Keller sp. nov. (Entomophthorales: Zygomycetes) infecting Tetraleurodes acaciae (Quaintance) (Insecta,Hemiptera: Aleyrodidae), a recently introduced whitefly on Gliricidia sepium (Jaq.) Walp. (Fabaceae) in the Philippines

Entomophthora leyteensis Villacarlos & Keller sp. nov., a species of Entomophthorales infecting the whitefly Tetraleurodes acaciae on Gliricidia sepium in the Philippines is described. Disease prevalence monitored weekly for 8 weeks indicated that the fungus could cause 8-31% infection within the whitefly population. Epizootics due to this fungus occurred in Inopacan, Leyte. Sampling live whitefly adults and dissecting them on glass slides for microscopic examination of fungal structures was found to give a better measure of prevalence than actual counts of infected insect cadavers. E. leyteensis is an important mortality factor for T. acaciae. Some speculations on the origin of the fungus are discussed here.     

Interaction and ovicidal activity of nematophagous fungus Pochonia chlamydosporia on Taenia saginata eggs

The ovicidal activity of the nematophagous fungi Pochonia chlamydosporia (isolates VC1 and VC4), Duddingtonia flagrans (isolate AC001) and Monacrosporium thaumasium (isolate NF34) on Taenia saginata eggs was evaluated under laboratory conditions. T. saginata eggs were plated on 2% water-agar with fungal isolates and controls without fungus and examined after 5, 10 and 15 days. At the end of the experiment P. chlamydosporia showed ovicidal activity against T. saginata eggs (p < 0.05), mainly for internal egg colonization with results of 12.8% (VC1) and 2.2% (VC4); 18.1% (VC1) and 7.0% (VC4); 9.76% (VC1) and 8.0% (VC4) at 5, 10 and 15 days, respectively. The other fungi showed only lytic effect without morphological damage to the eggshell. Results demonstrated that P. chlamydosporia was effective in vitro against T. saginata eggs unlike the other fungi.     

JM47, a cyclic tetrapeptide HC-toxin analogue from a marine Fusarium species

The known metabolite, enniatin B, and a cyclic tetrapeptide, JM47, which is a new natural product, were extracted from brown rice cultures of a marine fungus, identified as a Fusarium species, isolated from the marine alga Codium fragile. NMR studies, including 15N HMQC and 15N HMBC, established the structure of JM47 as cyclo(Ala-Ala-Aoh-Pro), where Aoh is the amino acid, (2S,9S)-2-amino-8-oxo-9-hydroxydecanoic acid. The absolute stereochemistry of the Aoh side chain carbinol centre was determined using Mosher ester methodology. Analysis of NOESY data assisted by molecular modelling revealed an alternating L-, D-, L-, D-configuration for the tetrapeptide core. The absolute stereochemistry of the core was determined by acidic hydrolysis and chiral TLC analysis of the proline residue. JM47 belongs to the HC-toxin family of cyclic tetrapeptides which possess a 2-amino-8-oxo-9,10-epoxydecanoic acid residue in place of the Aoh unit. This is the first report of an analogue of HC-toxin from a marine Fusarium species.     

Hirsutella thompsonii and Metarhizium anisopliae as potential microbial control agents of Varroa destructor,a honey bee parasite

The potential of Hirsutella thompsonii Fisher and Metarhizium anisopliae (Metschinkoff) as biological control agents of the parasitic mite, Varroa destructor Anderson and Trueman was evaluated in the laboratory and in observation hives. In the laboratory, time required for 90% cumulative mortality of mites (LT(90)) was 4.16 (3.98-4.42) days for H. thompsonii and 5.85 (5.48-7.43) days for M. anisopliae at 1.1 x 10(3) conidia mm(-2). At a temperature (34+/-1 degrees C) similar to that of the broodnest in a honey bee colony, Apis mellifera L., H. thompsonii [LC(90)=9.90 x 10(1) (5.86-19.35) conidia mm(-2) at Day 7] and M. anisopliae [LC(90)=7.13 x 10(3) (2.80-23.45) conidia mm(-2) at Day 7] both showed significant virulence against V. destructor. The applications of H. thompsonii to observation hives resulted in significant mortality of mites, and reduction of the number of mites per bee 21 and 42 days post-treatments. The treatments did not significantly affect the mite population in sealed brood. However, the fungus must have persisted because infected mites were still observed [82.97+/-(0.6)%] 42 days post-treatment. In addition, the fungus was found to sporulate on the host. A small percentage [2.86+/-(0.2)%] of dead mites found in the control hives also showed fungal infection, suggesting that adult bees drifted between hives and disseminated the fungus. H. thompsonii was harmless to the honey bees at the concentrations applied and did not have any deleterious effects on the fecundity of the queens. Microbial control with fungal pathogens provides promising new avenues for control of V. destructor and could be a useful component of an integrated pest management program for the honey bee industry.