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.     

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.     

Mycoviruses of Botrytis cinerea isolates from different hosts

Botrytis cinerea (teleomorph Botryotinia fuckeliana) is a necrotrophic plant pathogenic fungus that causes grey mould and enormous economic losses worldwide in different crops. Control of B. cinerea is difficult due to the appearance of fungicide‐resistant isolates, and the diversity in virulence due to genetic variability and, perhaps, the infection with mycoviruses or fungal viruses. The discovery of mycoviruses and their possible application as biocontrol agents, as well as their use as tools to study the plant–pathogen interaction, has encouraged their study in B. cinerea. Herein, we have analysed the occurrence of mycoviruses in Spanish B. cinerea isolates to approach a better understanding of the interactions among viruses, fungi and plants in this pathosystem. Fifty‐five percent of the B. cinerea isolates analysed contained double‐stranded RNA (dsRNA) elements, and the number of dsRNA elements, their relative concentration and size were variable among isolates. Some of these dsRNAs were related to the presence of virus like rod or isometric particles, and to cellular degeneration and malformed mitochondria. We have also demonstrated that a 3 kb dsRNA present in 55% of the isolates having dsRNA elements was a mycovirus genome. Partial sequence of that mycovirus presented high identity in nucleotide and amino acid sequence with Botrytis cinerea mitovirus 1 (BcMV1). Analysis of the genetic distance within Spanish BcMV1 sequences showed the existence of different isolates of this mitovirus inside the Spanish B. cinerea population analysed. This is the first report of the variability of dsRNA elements and the partial genome sequence of a mitovirus associated with Spanish B. cinerea isolates and the genetic diversity within Spanish isolates of BcMV1.     

Isolation and identification of secondary metabolites from hexane extract of culture filtrate of Bacillus licheniformis MTCC 7445

Hexane extract of cell free culture filtrate of Bacillus licheniformis MTCC 7445 isolated from rhizoshere soil of a resistant tomato plant showed antifungal activity against a number of soilborne and human pathogenic fungi. Maximum activity was observed against Botrytis cinerea (ED₅₀ = 23.79 μg/ml), Candida albicans (ED50 33.45 μg/ml) and Microsporum canis (ED₅₀ = 39.02 μg/ml). Metabolites such as 1-methyl pyrrolidene, 1-methyl cyclohexene, 4,4-dimethyl cyclohexane, ethyl-4-ethoxybenzoate, 2-butoxyethanol, naphthalene, ter butyl benzene and phenoxy acetic acid were identified by GC-MS and comparing the mass spectrum with the NIST library.     

Integrated management of faba bean chocolate spot caused by Botrytis fabae in Gondar,Ethiopia

Abstract

Integrated management of faba bean chocolate spot (Botrytis fabae Sard.) was studied using moderately tolerant variety, “Hachalu” and the local cultivar at Chilga district during 2016 main cropping season. The objective was to evaluate effect of host resistance, intercropping and fungicide applications on epidemics of faba bean chocolate spot, yield and yield components of faba bean, and sole and intercrop systems productivity. The experiment was randomised complete block design in a factorial combination of two faba bean cultivars, two cropping systems (Hachalu- and local-barley intercropping in 2:1 ratio) and four levels of fungicide (Chlorothalonil) spray and integration in three replications of faba bean and barley, respectively. Cultivars and spray intervals significantly (p?<?.01) affected the seed yield while cultivars alone had significant variation (p?<?.01) on 100-seed weight as a main effect. Therefore, 21-days fungicide spray intervals integrated with Hachalu sole and Hachalu-barley intercropping may be considered for chocolate spot management.     

Growth of Lesions Caused by Botrytis fabae on Field Bean Leaves in Relation to Foliar Bacteria, Non-enzymic Phytotoxins, Pectic Enzymes and Osmotica

Extracts from spreading chocolate spot lesions contained a heat-stable,water-soluble, ether- or ethanol-insoluble phytotoxic fraction.Elution from Sephadex G75 indicated that the molecular weightsof the toxic compounds were between 10000 and 30000 daltons.The toxins were adsorbed on DEAE Sephadex and eluted from itwith 0.1–0.2 M NaCl. Toxic activity was enhanced in solutionswith low osmotic potentials similar to those found in lesions.Controlling the growth of contaminating bacteria in lesionswith antibiotics appeared to reduce the levels of heat-stabletoxins extracted and to reduce the rate of increase in lesionsize. There were high levels of polygalacturonase (PG) activity inlesion extracts; large quantitites of PG, but little or no trans-eliminases,were produced by Botrytis fabae in liquid culture. A pectolyticstrain of Bacillus lentus was associated with trans-eliminasesin lesion extracts, and produced transeliminases, but not PG,in liquid culture. Activities of both heat-stable phytotoxins and of pectic enzymesmay depend on fungal isolate and types and populations of contaminatingbacteria. Botrytis fabae L., Bacillus, Vicia faba L., phytotoxins, pectic enzymes, chocolate spot     

Fungicide resistance of Botrytis cinerea in tomato greenhouses in the Canary Islands and effectiveness of non-chemical treatments against gray mold

Tomato greenhouses in the Canary Islands, Spain, were surveyed to estimate frequencies of resistance to benzimidazoles, dicarboximides, anilinopyrimidines and N-phenylcarbamates in Botrytis cinerea. Resistance to carbendazim, iprodione, pyrimethanil and diethofencarb was found in 74.2, 86.4, 28.8 and 31.8 % of isolates, respectively. Benzimidazole- and anilinopyrimide-resistant isolates were highly resistant, showing EC₅₀ values above 500 µg/ml carbendazim and a mean EC₅₀ value of 28.42 µg/ml pyrimethanil, respectively. By contrast, a low level of resistance was observed among dicarboximide-resistant isolates (mean EC₅₀ value of 1.81 µg/ml iprodione). Phenotypes with double resistance to carbendazim and iprodione, and triple resistance to carbendazim, iprodione and pyrimethanil were the most common, occurring in 36.4 and 28.8 % of isolates. The surveyed greenhouses had never been treated with fenhexamid and Signum? (pre-packed mixture of boscalid and pyraclostrobin), and baseline sensitivities of B. cinerea isolates to these fungicides were determined. The EC₅₀ values were within the range of 0.009–0.795 µg/ml fenhexamid and of 0.014–0.48 µg/ml Signum. In addition, available formulations based on elicitors of plant defense response and biocontrol agents were evaluated against B. cinerea in tomato plants under semi-controlled greenhouse conditions, the yeast Candida sake CPA-1 being able to reduce gray mold significantly when it was applied on petiole wounds and the plants were inoculated 24 h later. Likewise, C. sake was effective against B. cinerea in harvested tomato fruits, yeast-treated tomatoes showed a 70.66 and 30.31 % reduction in the diameters of decay lesions compared with controls after 10 days of storage at 20 and 9 °C, respectively.     

Yield loss of faba bean (Vicia faba) due to chocolate spot (Botrytis fabae) in sole and mixed cropping systems in Ethiopia

Two field experiments were conducted during 2004 and 2005 cropping seasons at Adet Agricultural Research Center, Ethiopia to assess yield losses caused by chocolate spot(Botrytis fabae) of faba bean in sole and mixed cropping systems using two cultivars. Cropping systems were sole faba bean (FB), faba bean mixed with field pea (FB: FP), barley (FB: BA) and maize (FB: MA). Mancozeb was sprayed at the rate of 2.5 kg a.i/ha at 7-, 14- and 21-day interval to generate different levels of chocolate spot disease in all the four cropping systems, and unsprayed control was also included. The treatments were arranged in a randomised complete block design (RCBD) with four replications. FB: MA mixed cropping significantly reduced disease severity and the area under disease progress curve (AUDPC) and increased faba bean grain yield. The highest faba bean grain yield among the three mixed croppings under different spray schedules was obtained from FB: MA mixed cropping in both 2004 and 2005 (2.56 and 3.74 t/ha, respectively) cropping seasons. There were highly significant yield differences (P < 0.05) among the spray intervals of mancozeb in both seasons. The highest grain yield (4.9 t/h) was recorded from the 7-day spray interval in 2005. The unsprayed faba bean had a lower grain yield (1.9 t/ha in 2004 and 2.3 t/ha in 2005) compared to the sprayed plots. The highest relative yield loss (67.5%) was calculated in 2005 from FB: FP mixed cropping in unsprayed plots. The relative yield losses in the unsprayed plots were in the range of 35.8–41.5% in 2004 and 52.6–67.5% in the 2005 cropping season. Severity and AUDPC were inversely correlated with faba bean grain yield. Significant differences were recorded in the 100-seed weight and days to maturity (DM). The unsprayed plots had shorter DM ranging from 126 to 128.5 day (except FB: MA mixed cropping) in 2004 and 122–123.9 days in 2005. In the sprayed plots DM was relatively longer than the unsprayed plots. A higher seed weight was recorded in the sole FB (56 g) and FB: MA (55 g) mixed cropping, and the lowest value of 100-seed weight was recorded from FB: FP (53 g) mixed cropping. The productivity of the mixed cropping evaluated by land equivalent ratio (LER) exceeded that of sole cropping. Faba bean grain yield was highly influenced by the severity of chocolate spot. The disease affects the DM, forcing early maturing of the plants.     

Comparison of Botrytis cinerea Populations Collected from Tomato Greenhouses in Northern Algeria

To estimate the genetic diversity and population structure for a better understanding of the spread of Botrytis cinerea, we genotyped with nine microsatellite markers 174 isolates collected from four greenhouses during three growing seasons in the region of Bejaia. Four of these isolates were detected as Botrytis pseudocinerea according to the allele size at locus Bc6. For all other isolates further studied, all loci were polymorphic, with the mean number of alleles per locus ranging from 2.77 to 5.22. Considerable genetic variability was detected in all subpopulations (D* > 0.87; Hnb > 0.40). Based on the standardized index of association analysis, significant but low levels of clonality occurred, not excluding the possibility of recombination (r_D = 0.07, P < 0.001). A total of 109 haplotypes were characterized among the isolates, few of which were shared between subpopulations. This, together with moderate genetic differentiation among subpopulations according to the geographical origin (0.080 < F_ST < 0.167), suggested a low level of inoculum exchange among greenhouses and little carry‐over of inoculum from one sampling season to the next. The importance of genetic structure of B. cinerea populations is discussed and should be taken into consideration for the management of grey mould.     

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.     

Inhibitory effects on microbial growth of Botrytis cinerea and Erwinia carotovora on potato using of a biopolymer chitosan at different molecular weights

The antimicrobial efficiency of chitosan at different molecular weights (5, 37, 57 and 290 kDa) against Botrytis cinerea and Erwinia carotovora on potato (Solanum tuberosum L.) was investigated. In vitro study showed that chitosan of 37 kDa was the most active against E. carotovora (minimum inhibitory concentration (MIC) = 950 mg/L), whereas 5 kDa chitosan was the most active against B.cinerea. Coating of potato tubers with 100, 250 and 500 mg/L significantly decreased the rate of weight loss and chitosan of 37 kDa showed the best effect. The in vivo antibacterial effect indicated that all treatments (500, 1000 and 2000 mg/L) significantly inhibited the growth of E. carotovora compared with the control. The lowest decay incidence was observed with 37 kDa chitosan. However, the antifungal activity against B. cinerea inoculated of leaves showed no decay incidence at 500 and 1000 mg/L with 57 kDa chitosan after 48 h.     

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.     

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.     

Endogene Hyphen bei Botrytis cinerea Pers.

Endohyphae in Botrytis cinerea Pers. Genesis, following differentiation and significance for pathogenesis The formation of endohyphae (secondary hyphae) in different cell types of Botrytis cinerea Pers. (6 isolates) is described. The following differentiation possibilities of these endohyphae within and—after egress—outside their cells of origin are presented. The influence of external factors (substrate, temperature, humidity, O₂-concentration) on these processes is considered. The significance of the endohyphae for pathogenesis is mainly due to their direct infectivity as well as the ability to develop normal mycelium.     

Effect of cultural methods on leaf spot (<Emphasis Type="Italic">Mycosphaerella fragariae</Emphasis>) and gray mold (<Emphasis Type="Italic">Botrytis cinerea</Emphasis>) damage in strawberries

Damage of leaf spot, caused by Mycosphaerella fragariae and gray mold also called Botrytis fruit rot, caused by Botrytis cinerea, average fruit weight and yield were evaluated with regard to cultural methods over 2years. Leaf spot damage decreased significantly by around 90% due to leaf sanitation (removal of dead and leaf spot infected leaves in early spring) and by 50% due to plantation in a one-row-system instead of a two-row-system. When all leaves including the healthy green ones were removed in early spring, average fruit weight decreased significantly by 10%. Fruit sanitation – the third treatment – did not influence any of the measured parameters. Neither leaf sanitation nor fruit sanitation (removal of damaged fruits during harvest) reduced B. cinerea damage significant. Only the combination of a one-row-system, leaf sanitation and fruit sanitation almost halved (not significantly) B. cinerea damage in the first crop year compared to a two-row-system without leaf and fruit sanitation. B. cinerea damage correlated significantly and positively with the biomass of plants by R²= 0.47. According to this study and the cited literature it is suggested for humid Central European conditions to apply a one-row-system combined with leaf sanitation in early spring and fruit sanitation during harvest if fruit density is high, to reduce the risk of damages in larger dimension caused by M. fragariae and B. cinerea.     

Synthesis and QSAR study of novel α-methylene-γ-butyrolactone derivatives as antifungal agents

Thirty-six new α-benzylidene-γ-lactone compounds based α-methylene-γ-butyrolactone substructure were prepared and characterized by spectroscopic analysis. All compounds were evaluated for antifungal activities in vitro against six plant pathogenic fungi and the half maximal inhibitory concentration (IC₅₀) against Botrytis cinerea and Colletotrichum lagenarium were investigated. Compounds 5c-3 and 5c-5 with the halogen atom exhibited excellent fungicidal activity against B. cinerea (IC₅₀ = 22.91, 18.89 μM). The structure-activity relationships (SARs) analysis indicated that the derivatives with electron-withdrawing substituents at the meta- or para-positions improves the activity. Via the heuristic method, the generated quantitative structure-activity relationship (QSAR) model (R² = 0.961) revealed a strong correlation of antifungal activity against B. cinerea with molecular structures of these compounds. Meanwhile, the cytotoxicity of 20 representative derivatives was tested in the human tumor cells line (HepG2) and the hepatic L02 cells line, the result indicated that the synthesized compounds showed significant inhibitory activity and limited selectivity. Compound 5c-5 has the highest fungicidal activity with IC₅₀ = 18.89 μM (against B. cinerea.) but low cytotoxicity with IC₅₀ = 35.4 μM (against HepG2 cell line) and IC₅₀ = 68.8 μM (against Hepatic L02 cell line). These encouraging results can be providing an alternative, promising use of α-benzylidene-γ-lactone through the design and exploration of eco-friendly fungicides with low toxicity and high efficiency.     

Resistenz verschiedener Kultur- und Wildtomatenpflanzen (Lycopersicon spp.) gegenüber Botrytis cinerea Pers.

Resistance of different cultivated and wild tomato plants (Lycopersicon spp.) to Botrytis cinerea Pers. 20 provenances of different cultivated and wild tomato plants (Lycopersicon spp.) were screened for resistance to Botrytis cinerea Pers. using an in vitro-leaf necrosis test. The Botrytis resistance decreased with increasing age of the leaves corresponding to their insertion height (relative youth resistance respectively senescence susceptibility). The 6 B. cinerea-isolates used for inoculation differed significantly in virulence. With increasing inoculum age a virulence reduction of the various B. cinerea-isolates occurred. Within the investigated test plant collection 2 wild species –L. columbianum and L. hirsutum– proved to be resistant in each stage of development to all B. cinerea-isolates and additionally showed field resistance.     

Brief in vitro study on Botrytis cinerea and Aspergillus carbonarius regarding growth and ochratoxin A

Aims: To evaluate the effect of Botrytis cinerea growth on ochratoxin A (OTA) production by Aspergillus carbonarius and degradation. Methods and Results: OTA‐producing A. carbonarius and B. cinerea were grown on grape‐like medium at 20°C for 7 days. Radii of colonies were daily recorded and OTA was analysed. In addition, each B. cinerea isolate was inoculated on grape‐like synthetic nutrient medium (SNM) paired with each A. carbonarius isolate at a distance of 45 mm. Botrytis cinerea isolates were also grown in OTA‐spiked SNM. Growth rates of B. cinerea and A. carbonarius were 20 and 7·5 mm day^?1, respectively. The growth of the colonies of each species stopped when they contacted each other in paired cultures. OTA production by A. carbonarius in the contact area was affected by B. cinerea, but no clear trend was observed. All B. cinerea isolates showed to degrade between 24·2% and 26·7% of OTA from spiked SNM. Conclusions: The ecological advantage of B. cinerea, in terms of growth rate, vs. OTA‐producing Aspergillus in some wine‐growing regions and its ability to degrade OTA may explain the low levels of this toxin in noble wines. Significance and Impact of the Study: At determinate conditions, the presence of B. cinerea in grapes with A. carbonarius may help in reducing OTA accumulation.     

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.     

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.