Changes in wyerone acid concentrations in leaves of Vicia faba after infection by Botrytis cinerea or B.fabae

Wyerone acid was produced by leaves of Vicia faba in response to infection by both Botrytis cinerea and B.fabae. Host cell death caused by either fungus appeared to be the trigger for rapid wyerone acid synthesis, although the phytoalexin was not confined to brown cells. At B. cinerea inoculation sites wyerone acid concentration increased rapidly, at the time of fungal invasion of the epidermis, to levels greater than that completely inhibitory to mycelial growth. Wyerone acid is therefore probably the primary cause of the inhibition of B. cinerea within infected tissue. The partial blackening of B.fabae inoculation sites and surrounding peripheral tissues was accompanied by an increase in wyerone acid. There followed a striking decrease as tissues became completely blackened and invaded by B.fabae. B.fabae appeared to metabolize wyerone acid and prevent its accumulation in invaded tissues. Mycelial growth of B. fabae was less sensitive to wyerone acid than was B. cinerea. The differing abilities of B.fabae and B. cinerea to spread from lesions after both have induced wyerone acid production probably depend on both their differing sensitivities to the phytoalexin and their abilities to metabolize it to less toxic products.     

Wyerone Acid Phytoalexin Synthesis and Peroxidase Activity as Markers for Resistance of Broad Beans to Chocolate Spot Disease

Infection of broad bean (Vicia faba L.) leaves with Botrytis fabae (Sard.) resulted in wyerone acid phytoalexin accumulation and increase in peroxidase activities in resistant and susceptible broad bean cultivars. Rapid wyerone acid accumulation was observed in leaves of resistant cultivars that reached levels greater than twofold of the susceptible cultivars. Following infection of broad bean cultivars, either resistant or susceptible to B. fabae, wyerone acid synthesis in resistant cultivars peaked at 5 days then declined, whereas in susceptible cultivars synthesis gradually decreased. While the ethanol‐extract of wyerone acid significantly reduced B. fabae spore germination with increased concentrations, the phytoalexin had no effect on in vitro B. fabae germ‐tube growth. Peroxidase activity in infected leaves of resistant was 10 times higher than the susceptible cultivars and eight times higher in uninfected leaves of the resistant than the susceptible cultivars. Peroxidase activity in infected leaves was greater than twofold in resistant cultivars and less than twofold in susceptible cultivars, when compared with uninfected leaves. The ratio of peroxidase activity in infected to uninfected leaves increased over time in susceptible cultivars but remained unchanged in resistant cultivars. Peroxidase activity in uninfected and infected leaves and wyerone acid biosynthesis in infected broad bean plants were successfully used to ascertain the resistance and susceptibility of four broad bean cultivars to B. fabae. Using wyerone acid synthesis and peroxidase activity as preliminary markers for resistance of broad bean to chocolate spot disease, caused by B. fabae, is suggested in this study.     

Biochemical changes in infection droplets containing spores of Botrytis spp. Incubated in the seed cavities of pods of bean (Vicia faba L.)

Infection droplets containing spores of Botrytis cinerea become inhibitory to the growth of germ tubes of the fungus within 18 hr. of their incubation in bean pods. The inhibition is caused by an ether-soluble substance, which has been partially purified, and which counteracts the stimulatory effect of sucrose, glucose, fructose, galacturonic acid and several amino acids, which are also present in the infection droplets. Changes in concentration of these substances have been described in the first 24 hr. after placing infection droplets in pods. The only major difference between droplets containing B. fabae and B. cinerea concerns the nature of the ether-soluble substances produced. Following B. fabae infection a biologically inactive u.v. absorbing substance appears in high yield in place of the antifungal substance formed following B. cinerea infections.     

The rates of fungal development and lesion formation in leaves of Vicia faba during infection by Botrytis cinerea and Botrytis fabae

Loss of the water droplet above inoculation sites during the first day after inoculation inhibited lesion formation by Botrytis cinerea and prevented the development of spreading lesions of B. fabae. With droplets present two general patterns of infection by B. cinerea were determined; in one, few or no symptoms were produced and in the other, limited lesions developed with marked browning of the inoculation site. Where few or no symptoms were produced, germination and germ-tube growth were inhibited on the leaf surface. B. cinerea was inhibited within the leaf at sites bearing limited lesions; invading hyphae were restricted to brown epidermal cells. Fungal growth on the leaf surface was greatest at sites with most browning beneath the droplet area. Variation in lesion development by B. cinerea could not be related to droplet position or leaf damage during normal preparation for inoculation. Plants differed in their susceptibility to lesion formation by B. cinerea. B. fabae, with droplet present, was not inhibited on the leaf surface and spread inter- and intra-cellularly beneath the inoculum drop and then into surrounding tissues. Delay in spread until the inoculation site was completely necrotic and colonized suggested that B. fabae is partially inhibited during the initial phase of infection. The rate of lesion spread varied in different plants and was most rapid in the youngest leaves.     

Interrelationship between Nutrients, Pathogenicity and Phytoalexin Metabolism of Botrytis cinerea on Clover Leaves

Botrytis cinerea spores suspended in 0.28 M glucose solution caused limited lesions on clover leaves, on which the clover phytoalexins maackiain and medicarpin accumulated to 1028 μg and 856 μg/g fresh wt respectively after 4 days incubation. During this time, little or none of the phytoalexin degradation products were detected. On the other hand, B. cinerea spores in sucrose casamino acids (SCA) liquid medium caused larger lesions than spores in glucose, and less maackiain and medicarpin (298 μg and 95 μg/g fresh wt respectively) and high concentrations of the degradation products were detected. B. cinerea mycelium in SCA also caused large lesions and both the phytoalexins and their degradation products were detected.,Sclerotinia laxa spores in 0.28 M glucose or its mycelium in SCA liquid medium did not cause any lesions apart from a minute necrotic fleck, and although phytoalexins were recovered from leaves inoculated with spores (67 μg and 78 μg/g fresh weight of maackiain and medicarpin respectively after 4 days) and leaves inoculated with mycelium (150 μg and 167 μg/g fresh wt maackiain and medicarpin respectively after 3 days), no phytoalexin degradation products were detected. The implications of, these results in understanding the interrelationship between nutrients, pathogenicity and phytoalexin metabolism are discussed.     

Phytoalexin Induction as a Factor in the Protection of Capsicum annuum L. Fruits Against Infection by Botrytis cinerea Pers.

Immature Capsicum annuum fruits treated at wound-sites with glucans from the hyphal walls of Glomerella cingulata and incubated for 24 h or longer accumulated levels of capsicannol phytoalexins sufficient to inhibit rot development by Botrytis cinerea. Elicitor application to intact fruits induced the accumulation of relatively low levels of capsicannol compounds but further phytoalexin accumulation in fruits after wound-inoculation with B. cinerea was much more rapid than in untreated fruits and rot development was suppressed. Capsicannol phytoalexins were produced in the live cells of the epidermis and little or no necrosis was associated with quantities produced in intact tissues. Elicitor treatment of intact fruits to protect against rot development by B. cinerea was achieved without the fruits sustaining unsightly damage.     

Conversion of wyerone to wyerol by Botrytis cinerea and B. fabae in vitro

The phytoalexin wyerone was induced to accumulate in cotyledons of Vicia faba infected with Botrytis cinerea or B. fabae. The acetylenic keto ester, wyerone, was converted to the less antifungal corresponding hydroxy ester, wyerol, by both species of Botrytis in vitro.     

Some Characteristics of Red Light-induced Substance(s) against Botrytis cinerea Produced in Broad Bean Leaflets

The red light-induced antifungal substance(s) produced in broad bean was of relatively high molecular weight, water soluble, heat stable and fungi specific. Cellulose thin layer chromatography (TLC) of infection droplets of Botrytis cinerea or water droplets without spores of B. cinerea, recovered from inoculated broad bean leaflets kept under red light for 48 h, displayed inhibition zones at approximate Rf values of 0.0 and 0.6. Inhibition zones observed in cellulose TLC of water droplets were relatively faint compared to those of infection droplets. In a time-course study of accumulation of the antifungal substance(s), antifungal activity in both water and infection droplets recovered from red light irradiated broad bean leaflets occurred after 24 h irradiation. However, the antifungal activity in infection droplets was significantly higher than in water droplets. The antifungal substance(s) was less active against Botrytis fabae than B. cinerea.     

The ABC transporter BcatrB from Botrytis cinerea exports camalexin and is a virulence factor on Arabidopsis thaliana

Arabidopsis thaliana is known to produce the phytoalexin camalexin in response to abiotic and biotic stress. Here we studied the mechanisms of tolerance to camalexin in the fungus Botrytis cinerea , a necrotrophic pathogen of A. thaliana . Exposure of B. cinerea to camalexin induces expression of BcatrB , an ABC transporter that functions in the efflux of fungitoxic compounds. B. cinerea inoculated on wild-type A. thaliana plants yields smaller lesions than on camalexin-deficient A. thaliana mutants. A B. cinerea strain lacking functional BcatrB is more sensitive to camalexin in vitro and less virulent on wild-type plants, but is still fully virulent on camalexin-deficient mutants. Pre-treatment of A. thaliana with UV-C leads to increased camalexin accumulation and substantial resistance to B. cinerea. UV-C-induced resistance was not seen in the camalexin-deficient mutants cyp79B2/B3 , cyp71A13 , pad3 or pad2 , and was strongly reduced in ups1 . Here we demonstrate that an ABC transporter is a virulence factor that increases tolerance of the pathogen towards a phytoalexin, and the complete restoration of virulence on host plants lacking this phytoalexin.     

Application of benzothiadiazole and Trichoderma harzianum to control faba bean chocolate spot disease and their effect on some physiological and biochemical traits

Chocolate spot disease is the most prevalent and important disease in the major faba bean growing regions in the world. Different concentrations of the abiotic inducer (0.3 and 0.5 mM benzothiadiazole) and the biotic inducer (1 × 10⁷ and 2 × 10⁷ spore/ml Trichoderma harzianum) were used alone or in combination to study their efficiency against faba bean chocolate spot disease caused by Botrytis fabae and Botrytis cinerea and their effect on some chemical analyses (phenylalanine ammonia lyase activity, total flavonoids and peroxidase isozymes, pectin and lignin content and total chlorophyll content). Application of the tested inducers as foliar treatment significantly reduced the severity of chocolate spot disease as compared with untreated infected plants. The reduction in disease severity was associated with a gradual increase in phenylalanine ammonia lyase activity. Maximum increase was recorded at 72 h after inoculation with B. fabae and B. cinerea. In addition, the levels of flavonoids in induced infected leaves recorded a sharp increase at 24 h after inoculation with B. fabae or B. cinerea. Also, pectin and lignin contents in the cell wall of induced infected plants were significantly increased as compared with untreated infected plants. Beside the induction of resistance, the tested inducers markedly increased total chlorophyll content in treated infected plants as compared with untreated infected plants. Isozymes analysis revealed that new peroxidase bands were induced only in treated faba bean leaves in response to infection with B. fabae or B. cinerea.     

Effects of Bean Leaf Age on Pathogenicity by Botrytis fabae

Leaves from field bean plants grown out of doors were inoculated with conidia of B. fabae immediately after detaching from stems. The oldest leaves developed more lesions than youngest ones, although they were not chlorotic. On intact plants at high humidities, established lesions on young leaves increased in size at only half the rate of those on old. but still green leaves. Seven days after inoculation a higher proportion of lesions on old leaves bore conidia than those on young leaves, but leaf age had no significant effect on numbers of conidia per mm² of lesion area. Young leaflets from bean plants grown in a controlled environment or in the field challenged with β. cinerea accumulated more phytoalexins than did old ones.     

The field behaviour of seed-borne Ascochyta fabae and disease control in field beans

In field sowings at Cambridge 2–15% of field bean seeds carrying Ascochyta fabae produced seedlings with leaf lesions. The fungus spread for distances up to 10 m in an average season and usually infected the new crop of seed. The amount of such infection arising from a single lot varied widely when samples were grown at different centres, presumably because of differences in local weather conditions. Seed lots with approximately 1% infected seeds seem suitable for ware crop production but little or no A. fabae can be tolerated in seed intended for multiplication. Infection in British-grown commercial seed has been greatly reduced by the selection of clean seed. Health standards adopted in the Field Bean Seed Scheme may have eliminated A. fabae from one cultivar.     

Interactions of crop density of field beans, abundance of Aphis fabae Scop., virus incidence and aphid control by chemicals

The number of Aphis fabae Scop. per plant and per acre developing on field beans (Vicia faba L.) was inversely related to seeding rate (i.e. plant density) except sometimes at very low rates; with equal numbers of plants per acre, fewer aphids developed on plants in rows 11 in. than 22 in. apart. Plots sown in mid-March with more than about 150,000 plants per acre were more attractive than less dense stands to colonizing alate A. fabae, but established colonies multiplied most on the sparsest and least on the densest plots. The number of plants per acre infected by pea leaf-roll virus was inversely related to planting density. There were more virus-infected plants on II in. than on 22 in. spaced rows-in contrast to the numbers of A. fabae. A single spray with demeton-methyl, timed to control A. fabae, did not significantly decrease virus incidence. Grain yields of sprayed plots were little altered by increasing the seed rate above a critical minimum, except in one year when the densest crops lodged. Increased yields from spraying were closely related to the numbers of A. fabae on unsprayed plots. Dense planting (more than 400,000 plants per acre) prevented or greatly decreased losses caused by A. fabae in unsprayed plots except in one year when the aphids were exceptionally abundant.     

Web-site selection strategies of linyphiid spiders in alfalfa: implications for biological control

Site-specific foraging can enhance the ability of generalist predators to provide effective and sustainable levels of pest control in agroecosystems. This can result from increased growth rates, higher population densities, and improved capture frequencies of pests at high prey density microsites. We tested the hypothesis that linyphiid spiders would exhibit microhabitat-specific web-site selection strategies in alfalfa. This was predicted to result in high prey densities at web-sites compared to paired non-web-sites through direct, or indirect, selection of prey-rich habitats. A total of 22,242 potential prey items were collected from mini-sticky traps located at 896 microsites. Web-centered mini-sticky traps on the ground, representative of Erigone autumnalis Emerton (Araneae: Linyphiidae) webs, captured similar numbers of potential prey as paired non-web-centered traps nearby. However, aerial sticky traps at web-sites of Bathyphantes pallidus (Banks) (Araneae: Linyphiidae) contained significantly more Diptera and Empoasca fabae (Harris) (Homoptera: Cicadellidae) than paired non-web-centered sticky-traps. Prey activity-densities also varied between web-sites of E. autumnalis and B. pallidus. Diptera were dominant at aerial microsites of B. pallidus whilst Collembola were abundant on ground-based traps of E. autumnalis. These results suggest that in alfalfa, the pressure for selecting prey-rich web-sites by erigonine spiders is low, but B. pallidus exhibits a selective web-location strategy targeted towards high quality dipteran prey. These sites also captured large numbers of E. fabae, a major pest of alfalfa, thus implicating aerial-based linyphiines as valuable predators in biological control.     

Occurrence of Didymella fabae,the Teleomorph of Ascochyta fabae,on Faba Bean Straw in Spain

Pseudothecia of Didymella fabae, the teleomorph of Ascochyta fabae, were first observed on faba bean (Vicia faba) debris in Spain during autumn 1995. Most pseudothecia were mature by December–February. The ascospores gave rise to typical cultures of A. fabae, and conidia from these cultures caused ascochyta blight symptoms on inoculated faba bean plants. Placing straw‐bearing pseudothecia over the plants to allow ascospore discharge also resulted in typical ascochyta blight symptoms. Pseudothecia maturity and discharge of ascospores from the infested faba bean straw overlapped with the vegetative stage of the faba bean crop, which occurs in southern Spain during winter as the crop is sown in autumn and harvested in spring. These observations indicate that ascospores may serve as primary inoculum for the disease.     

Production of the Phytoalexin Resveratrol by Grapes as a Response to Botrytis Attack Under Natural Conditions

Resveratrol (3,5,4′-trihydroxystilbene) is one of the stress metabolites produced by grapevines in response to fungal infection or injury. The distribution of this compound in limited lesions caused by Botrytiscinerea on ripe grape clusters in the vineyard has been examined. Resveratrol was present predominantly in the non-infected fruits close to the necrotic area. This localized response can help to arrest the spread of established B. cinerea lesions as long as climatic conditions are unfavourable to the pathogen. When conditions become favorable, i.e. after a mild and humid period rapidly spreading lesions are observed on fruits despite the continued increasing production of resveratrol in the grapes which can reach three- to five-fold the value found before rot intensification. Significant amounts of resveratrol can be found prior to any detectable lesions in fruits collected from apparently healthy clusters thus suggesting that phytoalexin response in initiated soon after the recognition of the pathogen by the plant. The involvement of one or more endogenous signals coming from either the fungus or the plant in the induction of host defence responses has also been discussed.     

Population studies on the active stages of the black bean aphid, Aphis fabae Scop., on its winter host Euonymus europaeus L

Autumn populations of Aphis fabae Scop, on the primary host Euonymus europaeus L. were little affected by natural enemies, most of which had begun to hibernate before the aphid populations developed. The size of the population in spring was usually determined by the number of overwintering eggs on a bush. The fundatrices hatched about 3–6 weeks before natural enemies became common. The growth of large A. fabae populations was first halted by the effects of intra-specinc competition, notably by the production and departure of emigrant alatae and by adult apterae reproducing more slowly. Later, natural enemies, especially Adalia 2-punctata (L.), Syrphidae and the parasite Trioxys sp. (near angelicae), multiplied and accelerated the decline in the aphid populations, which usually disappeared in June leaving many immature natural enemies. Larval A. 2-punctata began to eat parasitized aphids and cannibalized other larvae and pupae. The small populations of A. fabae that develop from few overwintering eggs are at greater risk from natural enemies than are large ones. Intraspecific competition still slowed population increase, because most aphids remained crowded on the few originally colonized twigs. Such populations produced very few emigrant alatae before they were exterminated by the combination of T. angelicae with specific and non-specific predators. Adult Cantharidae killed many of the aphids, especially in hedgerow habitats, where they were abundant. Coccinellidae, Anthocoridae and syrphid larvae, and the adults of nonspecific predators, notably Cantharidae, prevented recolonization of E. europaeus throughout July and August. Leaves of E. europaeus may remain physiologically suitable for A. fabae throughout July but begin to deteriorate in August when A. fabae kept on them become less fecund. Experiments using exclusion techniques provided evidence that natural enemies which attack A. fabae on E. europaeus and on summer hosts cause the common 2-year cycle of aphid abundance. Individual E. europaeus differ consistently in the extent to which they are colonized by A. fabae. Conditions are discussed that should govern the choice of E. europaeus bushes on which the A. fabae populations can be used as sensitive indicators of later crop infestations.     

Factors affecting the relative abundance of two coexisting aphid species on sugar beet

1 The two most common species of aphid colonizing sugar beet Beta vulgaris L. are Myzus persicae (Sulzer) (Hemiptera: Aphididae) and Aphis fabae Scopoli (Hemiptera: Aphididae). 2 M. persicae colonizes sugar beet earlier than A. fabae but the population of the former also declines earlier. Despite similar numbers of each species migrating at the time of colonization, M. persicae is usually less abundant on the crop than A. fabae, suggesting differences between the species in their selection of, and performance on, sugar beet. 3 The intrinsic rate of increase of both species declines as sugar beet matures, however, at any given plant age the intrinsic rate of increase of A. fabae is one and a half times greater than that of M. persicae. This results in more rapid population growth and a later decline of the population. 4 Intraspecific competition appears to result in M. persicae becoming very restless, but there is no evidence for interspecific competition between the two species on this host. 5 A population growth model which takes account of the decline in host quality of sugar beet shows that the M. persicae population peaks 30 days before that of A. fabae, and, excluding differences in emigration rate, the maximum A. fabae population is 14 times greater than the maximum M. persicae population. These results are compared to field data.     

Tunisian isolates of Trichoderma spp. and Bacillus subtilis can control Botrytis fabae on faba bean

Faba bean crops worldwide are often attacked by different diseases, particularly chocolate spot caused by Botrytis fabae Sard. Fungal and bacterial isolates collected from faba bean and barley leaves in Tunisia were evaluated for their antagonistic potential against B. fabae. In a test on detached leaves, the highest rate of decrease in disease severity was scored by Trichoderma viride, followed by T. harzianum, the fungicide Carbendazim then Bacillus subtilis. Under glasshouse conditions, all tested fungi resulted in significant disease severity reduction. T. viride reduced the rate of chocolate spot infestation on leaves and stems by 35% and 31.5%, respectively, when the rate on the control was 100%. For T. harzianum, Carbendazim and B. Subtilis, the rates of infestation on the leaves were 41.7%, 43.1% and 59.7%, respectively. On the stems, T. harzianum scored the lowest rate of 54.2% followed by B. subtilis with 79.2% then Carbendazim with 87.5%. Two consecutive seasons of field trials using the Trichoderma species, B. subtilis and Carbendazim showed significant and consistent reduction in the severity of chocolate spot infestation rates. The highest protection against the disease was obtained from T. viride. Based on these results, Tunisian isolates of Trichoderma spp. can be recommended for developing commercial bio-fungicides for integrated management of chocolate spot.