Antagonistic interactions between fungal pathogens and phylloplane fungi of guava

Dominant phylloplane fungi of guava (Psidium guajava L.) were screened for their antagonistic activities against the two pathogens,Colletotrichum gloeosporioides andPestalotia psidii, bothin vitro andin vivo. Culture filtrates ofAspergillus niger, Fusarium oxysporum andPenicillium citrinum caused more than 50% growth inhibition ofC. gloeosporioides. Filtrates ofCephalosporium roseo-griseum andF. oxysporum were most effective in reducing the growth ofP. psidii. Volatiles produced from the cultures ofA. niger, F. oxysporum, P. citrinum andP. oxalicum inhibited the growth ofC. gloeosporioides, whereas volatiles fromC. roseo-griseum, F. oxysporum andTrichoderma harzianum inhibited the growth ofP. psidii. The inhibitory effect of volatiles decreased with increase in incubation time. In general, the maximum effect of volatiles was noticed after 48 h incubation. Different grades of colony interactions in dual cultures were recognised between the two pathogens and the phylloplane fungi examined. Maximum inhibition ofC. gloeosporioides was caused byAureobasidium pullulans, Cladosporium cladosporioides, epicoccum purpurascens, F. oxysporum andMyrothecium roridum, whereasAspergillus terreus, C. roseo-griseum andP. oxalicum significantly reduced the growth ofP. psidii. Application of a spore suspension of each test fungus inhibited lesion development of guava leaves caused by the test pathogensin vitro. Inhibition was more pronounced when the spore concentration was increased.A. pullulans, C. cladosporioides, E. purpurascens, F. oxysporum, andT. harzianum were found to be strongly antagonistic toC. gloeosporioides. A. niger, A. terreus, C. roseo-grisem andT. harzianum were strongly antagonistic toP. psidii.     

Exogenous double-stranded RNA inhibits the infection physiology of rust fungi to reduce symptoms in planta

Rust fungi (Pucciniales) are a diverse group of plant pathogens in natural and agricultural systems. They pose ongoing threats to the diversity of native flora and cause annual crop yield losses. Agricultural rusts are predominantly managed with fungicides and breeding for resistance, but new control strategies are needed on non-agricultural plants and in fragile ecosystems. RNA interference (RNAi) induced by exogenous double-stranded RNA (dsRNA) has promise as a sustainable approach for managing plant-pathogenic fungi, including rust fungi. We investigated the mechanisms and impact of exogenous dsRNA on rust fungi through in vitro and whole-plant assays using two species as models, Austropuccinia psidii (the cause of myrtle rust) and Coleosporium plumeriae (the cause of frangipani rust). In vitro, dsRNA either associates externally or is internalized by urediniospores during the early stages of germination. The impact of dsRNA on rust infection architecture was examined on artificial leaf surfaces. dsRNA targeting predicted essential genes significantly reduced germination and inhibited development of infection structures, namely appressoria and penetration pegs. Exogenous dsRNA sprayed onto 1-year-old trees significantly reduced myrtle rust symptoms. Furthermore, we used comparative genomics to assess the wide-scale amenability of dsRNA to control rust fungi. We sequenced genomes of six species of rust fungi, including three new families (Araucariomyceaceae, Phragmidiaceae, and Skierkaceae) and identified key genes of the RNAi pathway across 15 species in eight families of Pucciniales. Together, these findings indicate that dsRNA targeting essential genes has potential for broad-use management of rust fungi across natural and agricultural systems.     

Broad spectrum management of plant diseases by phylloplane microfungal metabolites

Plant surfaces are colonised by an array of microbes. These microbes have been implicated in protecting plants against invading pathogens. Studies have demonstrated that metabolites of phylloplane microfungi can induce defence responses in their host plants. This investigation relates to formulation of a biocide from metabolites of Aspergillus niger, Trichoderma viride, Alternaria alternata and Chaetomium globossum, which are common phylloplane colonisers of tropical plants. The biocide was found to reduce disease incidence through induction of alterations in defence physiology of crop plants.     

Toxicity of Essential Oil from a New Strain of Ocimum gratissimum (Clocimum) against Betelvine Pathogenic Fungi

The leaves of Ocimum gratissimum (Clocimum) exhibited strong volatile fungitoxicity against betelvine (Piper betle L.) pathogens—Alternaria alternata, Colletotrichum capsici and Sclerotium rolfsii. Fifteen compounds could be identified from the fungitoxic constituents—the essential oil. The oil at its minimum inhibitory concentrations of 50, 250 and 500 ppm against S. rolfsii, A. alternata and C. capsici, respectively, was fungistatic, although, fungicidal at higher concentrations. Eugenol was found to be the major fungitoxic principle in the oil. The oil was either equally effective or superior to synthetic commercial fungicides and was non-phytotoxic to the host plants. Thus, the oil can be used as a valuable indigenous and biodegradable agent against fungi that cause losses to the betelvine industry.     

Biocontrol potential of phylloplane bacterium Ochrobactrum anthropi BMO‐111 against blister blight disease of tea

Aims

The present study was carried out to screen the phylloplane bacteria from tea for antagonism against grey blight caused by Pestalotiopsis theae and blister bight caused by Exobasidium vexans and to further evaluate the efficient isolates for disease control potential under field condition.

Methods and Results

A total of 316 morphologically different phylloplane bacteria were isolated. Among the antagonists, the isolates designated as BMO‐075, BMO‐111 and BMO‐147 exhibited maximum inhibitory activity against both the pathogens under in vitro conditions and hence were selected for further evaluation under microplot field trial. Foliar application of 36‐h‐old culture of BMO‐111 (1 × 10⁸ colony‐forming units ml^?1) significantly reduced the blister blight disease incidence than the other isolates. The culture of BMO‐111 as well as its culture filtrate effectively inhibited the mycelial growth of various fungal plant pathogens. The isolate BMO‐111 was identified as Ochrobactrum anthropi based on the morphological and 16S rDNA sequence analyses.

Conclusions

It could be concluded that the biocontrol agent O. anthropi BMO‐111 was effective against blister blight disease of tea.

Significance and Impact of the Study

Further study is required to demonstrate the mechanism of its action and formulation for the biocontrol potential against blister blight disease of tea.     

Phylloplane microfungal metabolite influences activity of RuBisCO

The phylloplane microbes are known to affect systemic acquired resistance in plants against a broad spectrum of pathogens. The influence of phylloplane microbes and their metabolites on physiology and biochemistry of plants is well-documented but their effect on the physiology and functioning of chloroplasts and mitochondria is not well understood. The present study was conducted to understand the impact of metabolite of a dominant phylloplane microfungi (Fusarium oxysporum) isolated from the phylloplane of Lycopersicon esculentum on activity of RuBisCO. Activity of RuBisCO (EC 4.1.1.39) in chloroplasts of fungal metabolite treated and untreated eight-week old tomato plants were studied. Significant increase in the activity of RuBisCO (P???0.01), isolated from plants inoculated with the fungal metabolite was observed. The fungal metabolite could elicit the enzyme activity by about two folds (P???0.05) within 72–96?h of treatment of tomato plants. The result demonstrates that probably phylloplane microfungi and/or their metabolites influence the photosynthetic efficiency of plants.     

Apyrase inhibitors enhance the ability of diverse fungicides to inhibit the growth of different plant‐pathogenic fungi

A previous study has demonstrated that the treatment of Arabidopsis plants with chemical inhibitors of apyrase enzymes increases their sensitivity to herbicides. In this study, we found that the addition of the same or related apyrase inhibitors could potentiate the ability of different fungicides to inhibit the growth of five different pathogenic fungi in plate growth assays. The growth of all five fungi was partially inhibited by three commonly used fungicides: copper octanoate, myclobutanil and propiconazole. However, when these fungicides were individually tested in combination with any one of four different apyrase inhibitors (AI.1, AI.10, AI.13 or AI.15), their potency to inhibit the growth of five fungal pathogens was increased significantly relative to their application alone. The apyrase inhibitors were most effective in potentiating the ability of copper octanoate to inhibit fungal growth, and least effective in combination with propiconazole. Among the five pathogens assayed, that most sensitive to the fungicide‐potentiating effects of the inhibitors was Sclerotinia sclerotiorum. Overall, among the 60 treatment combinations tested (five pathogens, four apyrase inhibitors, three fungicides), the addition of apyrase inhibitors increased significantly the sensitivity of fungi to the fungicide treatments in 53 of the combinations. Consistent with their predicted mode of action, inhibitors AI.1, AI.10 and AI.13 each increased the level of propiconazole retained in one of the fungi, suggesting that they could partially block the ability of efflux transporters to remove propiconazole from these fungi.     

Fungal microflora of seeds of Pisum sativum L. and its control

Five varieties ofPisum sativum L. were tested for seedborne fungi. Tests were conducted by standard International Seed Testing methods. It was observed that fungi likeAlternaria, Aspergillus, Rhizopus, Mucor andFusarium were associated with all the five varieties tested.Fusarium andRhizopus were dominant in all the varieties. They were also responsible for reduction in germination percentage of seeds. Early December was selected for testing the efficacy of certain fungicides (namely, Agrosan G.N., Ceresan, Copper carbonate, Tafasan, Tillex, Stardex and Sulphur) against seedborne fungi ofPisum sativum L., Agrosan G.N. and Ceresan gave the better control of seedborne pathogens without any adverse effect on germination.     

Effect of Azotobacter Exudates on Certain Micro-Fungi Associated with the Phylloplane of Soybean

The effect of different concentrations of Azotobacter chroococcum exudates on microfungi isolated from Soybean phylloplane was studied in vitro and in vivo. It has been found that the growth of some fungi was stimulated or suppressed whereas others showed fluctuations or remained unaffected depending upon the age of the plant, growth stage of the leaf, concentration of the exudates, method used to harvest fungi, and environmental conditions. Nutrient competition or production of biologically active substances like GA may be responsible for retarding the growth of fungi.     

Negatively Correlated Cross-resistance and Synergism between Phosphoramidates and Phosphorothiolates in Their Fungicidal Actions on Rice Blast Fungi

Alkyl aryl esters, dialkyl esters and diaryl esters of N-methyl-N-phenylphosphoramidic acid were synthesized and tested for cross-resistance and joint action with phosphorothiolate (PTL) fungicides using rice blast fungi, Pyricularia oryzae. Negative correlation in crossresistance was found between PTL fungicides and most of the amidates. Synergism in the fungicidal action was also found between them for wild types of the fungi, when tested by crossing filter paper strips impregnated with fungicides on agar plates inoculated uniformly with the test fungi.     

The phylloplane microflora of ripening wheat and effect of late fungicide applications

Flag leaves and ears of spring wheat cv. Timmo (in 1980) and winter wheat cv. Maris Huntsman in 1981 and 1982 were colonised by a variety of micro-organisms whose numbers increased rapidly between anthesis and harvest. The predominant mycoflora were yeasts, yeast-like fungi and filamentous fungi which included Cladosporium spp., Verticillium lecanii, Alternaria alternata, Fusarium spp. and Epicoccum nigrum. Although similar species were isolated, their relative abundance on flag leaves and ears differed. The fungicide captafol was most effective as a protectant and significantly decreased populations of fungi on flag leaves and ears for 6 and 4 wk respectively, compared to untreated controls. Benomyl and Delsene M (carbendazim + maneb) were the most effective of the systemic sprays and formulations. In general, fungicides affected populations of yeasts, yeast-like fungi and Cladosporium spp. most while Alternaria was tolerant of all treatments. Yields of winter wheat were increased in two seasons by an average 0–2 t ha^-1 (2–4%) following a single late fungicide treatment at G.S. 50 or 60 and 0–41 t ha^-1 (5-1%) when this was combined with an early spray against foliar diseases (G.S. 38–40). Individual treatments increased yield by up to 12% with little difference between applications at G.S. 50 or 60. The yield benefit came mainly from increased 1000-grain weights. Germination of the treated grain was increased only slightly.     

Effect of fungicidal essential oils against Botrytis cinerea and Rhizopus stolonifer rot fungus in vitro conditions

The development of natural crop protection products as alternatives to the use of synthetic fungicides is currently popular. The aim of this study is to evaluate the antifungal effects of several essential oils against the fungal pathogens, Botrytis cinerea and Rhizopus stolonifer, under in vitro condition. Four essential oils (fennel, black caraway, peppermint and thyme) were each tested at five concentrations (0, 200, 400, 600 or 800 μl l^?1). In vitro results showed that the essential oil of black caraway and fennel had the highest fungicidal effect against B. cinerea and R. stolonifer, respectively. The growth of B. cinerea was completely inhibited by the essential oil of black caraway at 400 μl l^?1. Fennel oil perfectly inhibited growth of R. stolonifer fungus colonies at concentration higher than 600 μl L^?1 in potato dextrose agar medium. Percentage of spores germination was the lowest in medium of Fennel and black caraway essential oils, and was the highest in Thyme ones. These results show that plant essential oils can have a strong effect on reducing post-harvest decay. These plant essential oils could provide an alternative to synthetic chemicals to control post-harvest phytopathogenic fungi on fruit.     

Exploiting medicinal plants and phylloplane microflora for the management of grapevine downy mildew

Abstract

Grapevine downy mildew is the most devastating disease throughout the world causing huge monetary losses. Twenty medicinal plant extracts and six phylloplane microfloras were evaluated for their efficacy against sporangial germination of grapevine downy mildew pathogen Plasmopara viticola in vitro. The results revealed that the Neem Seed Kernel Extract (NSKE) at 5% significantly inhibited the sporangial germination (75.36%) of P. viticola. Among the phylloplane microflora Pseudomonas fluorescens was highly effective in reducing the sporangial germination (64.26%). Post inoculation spraying of NSKE (5%) and P. fluorescens (0.2%) effectively inhibited the disease development in the greenhouse. Three sprays with NSKE (5%) and phylloplane P. fluorescens (0.2%): first spray after initial appearance of disease and the second and third at 10 day intervals were found to be promising in reducing disease incidence in the field.     

Contribution of Pathogens to Peach Fruit Rot in Northern Greece and their Sensitivity to Iprodione, Carbendazim, Thiophanate-methyl and Tebuconazole Fungicides

This study identified the main pathogens causing fruit rots of mature peaches in northern Greece, the major peach producing area of Greece. The brown rot pathogen Monilinia laxa was responsible for approximately 70% and 78% of rotted peaches in 2005 and 2006 respectively. Serious damage (up to 5%) was also caused with the fungus Phomopsis amygdali. Other pathogens isolated from rotted peaches at a low percentage were Alternaria alternata, Aspergillus niger, Aspergillus flavus, Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium spp., Colletotrichum gloeosporioides, Rhizopus stolonifer and Gilbertella persicaria. Most fungal isolates originated from the rotted peaches were tested for their sensitivity to the fungicides iprodione, carbendazim, thiophanate methyl and tebuconazole at label recommended concentrations. All fungicides inhibited the growth of M. laxa, A. niger, A. flavus, S. sclerotiorum, P. amygdali and B. cinerea on poisoned agar. Apart from iprodione, all other fungicides inhibited the mycelium growth of the pathogen Fusarium sp. The mycelium growth of Fusarium sp. was significantly less with iprodione than control. Only iprodione and tebuconazole were effective against A. alternata and R. stolonifer. Tebuconazole inhibited the mycelium growth of R. stolonifer, while iprodione reduced significantly in comparison to control. The mycelium growth of the fungus C. gloeosporioides was inhibited by tebuconazole and reduced significantly by the fungicides thiophanate methyl, carbendazim and iprodione. Among all the fungi tested, only M. laxa and B. cinerea isolates were found resistant to benzimidazoles [the EC50 (50% effective concentration) value was 100–200 mg/l and 200–300 mg/l for the largest number of thiophanate methyl‐ and carbendazim‐resistant M. laxa isolates respectively, while the biggest number of B. cinerea thiophanate methyl‐ and carbendazim‐resistant isolates showed EC50 value 200–300 mg/l and 300–400 mg/l, respectively]. However, these strains were sensitive to tebuconazole and iprodione. Therefore, these fungicides can be used as an alternative method to control benzimidazole‐resistant Monilinia and Botrytis isolates.     

Chlorine Dioxide for Reduction of Postharvest Pathogen Inoculum during Handling of Tree Fruits

Alternatives to hypochlorous acid and fungicides are needed for treatment of fruit and fruit-handling facilities. Chlorine dioxide was evaluated and found effective against common postharvest decay fungi and against filamentous fungi occurring on fruit packinghouse surfaces. In vitro tests with conidial or sporangiospore suspensions of Botrytis cinerea, Penicillium expansum, Mucor piriformis, and Cryptosporiopsis perennans demonstrated >99% spore mortality within 1 min when the fungi were exposed to aqueous chlorine dioxide at 3 or 5 μg · ml^-1. Longer exposure times were necessary to achieve similar spore mortalities with 1 μg · ml^-1. Of the fungi tested, B. cinerea and P. expansum were the least sensitive to ClO₂. In comparison with the number recovered from untreated control areas, the number of filamentous fungi recovered was significantly lower in swipe tests from hard surfaces such as belts and pads in a commercial apple and pear packinghouse after treatment of surfaces with a 14.0- to 18.0-μg · ml^-1 ClO₂ foam formulation. Chlorine dioxide has desirable properties as a sanitizing agent for postharvest decay management when residues of postharvest fungicides are not desired or allowed.     

In vitro efficacy of some fungicides on mycelial growth of Alternaria alternata and Mucor pyriformis

Various chemical fungicides, systemic and non-systemic, were tested against fruit rot pathogens viz. Alternaria alternata and Mucor pyriformis for the evaluation of inhibition of mycelial growth. In A. alternata, among the systemic fungicides used, hexaconozole showed highest inhibition of mycelial growth followed by carbendazim and least effective was myclobutanil. While in M. pyriformis, hexaconozole showed highest inhibition and least effective was bitertanol. Among the non-systemic fungicides tested in both A. alternata and M. pyriformis, mancozeb showed highest inhibition of mycelial growth followed by capton and the least inhibition was shown by zineb.     

The microflora of ripening barley grain and the effects of pre harvest fungicide application

Newly-emerged ears of barley soon became colonised by micro-organisms with numbers increasing to 5·8 × 10⁵ viable propagules/g dry weight by harvest. Bacteria were numerically predominant throughout. Yeasts and yeast-like fungi were also numerous during the early stages of grain development but were exceeded by propagules of filamentous fungi during ripening, with Cladosporium spp., Alternaria alternata, Epicoccum purpurascens and Verticillium lecanii most abundant. Numbers of most micro-organisms were greatest in a wet season but Alternaria was equally abundant in hot dry weather. Application of fungicides at or after anthesis modified the grain microflora. Captafol was most effective overall in decreasing numbers of fungi. However, benomyl decreased Cladosporium inoculum more than catpafol but was ineffective against Alternaria which became more numerous on benomyl-treated grain than on untreated. Yield increases of up to 4% were obtained by late fungicide treatment, least in a hot, dry year. Germination was also increased by some treatments by up to 5%.     

Combining vegetable oil and sub-lethal concentrations of Imidacloprid with Beauveria bassiana and Metarhizium anisopliae against adult guava weevil Conotrachelus psidii (Coleoptera: Curculionidae)

Of the insect pests that attack guava fruits, the guava weevil, Conotrachelus psidii Marshall, 1922 (Coleoptera: Curculionidae), is one of the most important in Brazil. In search of alternatives to chemical pesticides, this study was performed to select fungal isolates of Beauveria bassiana and Metarhizium anisopliae as potential candidates for the control of adult C. psidii. Tests were carried out using three products applied with the entomopathogenic fungi: Tween 80, sunflower oil and Imidacloprid (IMI). A sub-lethal concentration of IMI was determined (100 ppm). The results demonstrated that LPP 19 and LPP 114 were the most effective isolates when used in combination with all of the products. The least virulent isolate, ESALQ 818, when applied in Tween 80 caused only 26.6% mortality, however, this isolate showed significantly improved efficiency when applied together with either sunflower oil or IMI, causing 57.3 and 88.6% mortality, respectively. The efficiency of all the isolates tested here improved when applied together with IMI, with LT₅₀ values of 5.3–10.3 days when compared to LT₅₀ values in Tween alone of 9.5–17 days. The isolate that produced the highest number of conidia on the cadavers of adult C. psidii was LPP138, independent of the product used; however, conidial production was slightly reduced when fungi were applied together with IMI. These results are promising for developing new formulations of the isolates to be tested in the field.     

Screening of bacteria for the suppression of Botrytis cinerea and Rhizoctonia solani on lettuce (Lactuca sativa) using leaf disc bioassays

Two leaf disc bioassays were developed for screening bacteria as putative biological control agents of Botrytis cinerea and Rhizoctonia solani on lettuce. Aerobic spore and non‐spore forming bacteria were isolated from the phylloplane, rhizoplane and rhizosphere of symptom‐free lettuce plants grown in the presence and absence of chitin or composted bark soil amendments. Bacteria, previously isolated from other plants, were also included in the primary screen initially against B. cinerea. One hundred and twenty‐seven of 700 isolates reduced botrytis rotting of lettuce leaves by more than 50% in the primary screen. Following a secondary screen against B. cinerea, the lead 50 isolates were also tested for suppression of R. solani infection. Four isolates significantly reduced both botrytis and rhizoctonia leaf rotting. Eleven and five isolates gave control of botrytis and rhizoctonia, respectively, equal to that given by the standard fungicides Rovral WP (iprodione) and Basilex (tolclofos methyl). The two most effective isolates against B. cinerea and R. solani were both identified as Bacillus subtilis. Use of soil amendments did not increase the proportion of efficacious isolates recovered. Effective isolates were originally recovered from roots of oilseed rape and lettuce leaves. In general, it was found that bacteria which controlled one disease effectively did not control the second disease nearly as well. The bioassay protocols developed in this study were used successfully in screening a large number of bacterial isolates in a short time.     

Efficacy of benzimidazole and related fungicides against Rhizoctonia solani and R. bataticola

Five formulations of four benzimidazole derived fungicides, carbendazim, benomyl, thiophanate methyl and methyl 4-[2-(2-dimethylamino acetamide) phenyl]-3-thioallophanate were compared for their toxicity towards two pathogenic isolates of Rhizoctonia solani and three of R. bataticola. The isolates of two fungi showed significant differences in mycelial growth inhibition by the five fungicides. Benomyl and carbendazim were most inhibitory to all isolates of both fungi while the sesame isolate of R. bataticola was least sensitive to all fungicides. Disease control (90%) was obtained with low concentrations of benomyl against root rot of cowpea caused by R. solani, and with thiophanate methyl against root rot of sesame and sunflower, and leaf blight of mung bean caused by R. bataticola. The spread of stalk-end rot of sunflower heads was best checked with a spray of thiophanate methyl. The results suggest that benzimidazole fungicides having similar toxophores act differently for disease control in different host-parasite combinations.