Possible explanations of synergism in fungicide mixtures against Phytophthom infestans

Single foliar sprays of oxadixyl, mancozeb or cymoxanil alone or the mixtures oxadixyl/mancozeb and oxadixyl/cymoxanil were applied to tomato plants (cv. Baby) one day before inoculating with a sensitive or a phenylamide-resistant strain of Phytophthora infestans. Fungicide mixtures were far more effective in controlling both sensitive and resistant strains compared to the individual components alone, representing significant levels of synergistic interactions. When analysed by the Abbott method, synergy ratios continuously decreased with increasing amounts of fungicides in the mixture. All fungicide mixtures showed higher synergy ratios against resistant strains when using the Abbott method, whereas only mixtures containing cymoxanil showed this phenomenon when using the Wadley method for interpretation of interactions. Synergism significantly decreased when the components were used as foliar split applications with intervals of 48 and 72 h. A combination of a drench application of oxadixyl and a single spray application of mancozeb or cymoxanil resulted in a high level of synergism. The explanation of synergism may be that the exposure of sporangia to sublethal concentrations of one fungicide affects them to an extent that sublethal doses of the other fungicide becomes detrimental.     

Disease Development and Buildup of Resistance to Oxadixyl in Potato Crops Inoculated with Phytophthora infestans as Affected by Oxadixyl and Oxadixyl Mixtures: Experimental and Simulation Studies

Simulator predictions for the effect of oxadixyl and oxadixyl mixtures on the control of late blight in potato crops inoculated with Phytophthora infestans and the buildup of fungal resistance to oxadixyl were compared with data obtained in field experiments. The simulator, which considered fungus, fungicides and crop variables, predicted accurately the effect of fungicide sprays (oxadixyl, oxadixyl mixed with mancozeb, and oxadixyl mixed with mancozeb and cymoxanil) on blight development. The model predicted accurately both resistance buildup and infection in non-treated crops and in crops treated with oxadixyl alone or in a mixture with mancozeb (SAN 518F), but overestimated the control efficacy of the three-way mixture Pulsan.     

Activity of Ten Fungicides against Phytophthora capsici Isolates Resistant to Metalaxyl

Pepper Phytophthora blight (PPB), caused by Phytophthora capsici, is an important disease of pepper in China. The extensive application of metalaxyl has resulted in widespread resistance to this fungicide in field. This study has evaluated the activities of several fungicides against the mycelial growth and sporangium germination of metalaxyl‐sensitive and metalaxyl‐resistant P. capsici isolates by determination of EC₅₀ values. The results showed that the novel carboxylic acid amide (CAA) fungicide mandipropamid exhibited excellent inhibitory activity against PPB both in vitro and in vivo, with averagely EC₅₀ values of 0.075 and 0.004 μg/ml in mycelial growth and sporangium germination, respectively, and over 88% efficacy in controlling PPB. The other three CAA fungicides also provided over 70% efficacy in controlling PPB. The mycelial growth was less sensitive to quinone outside inhibitor (QoI) fungicides azoxystrobin and trifloxystrobin than that of sporangium germination in P. capsici isolates. However, azoxystrobin and trifloxystrobin provided over 80% efficacy in controlling PPB. It was noted that propamocarb and cymoxanil did not exhibit activity against the mycelial growth or sporangium germination of P. capsici isolates in the in vitro tests, with over 70% efficacy in controlling PPB. The new fungicide mixture 62.5 g/l fluopicolide + 625 g/l propamocarb (trade name infinito, 687.5 g/l suspension concentrate (SC)) produced over 88% efficacy in controlling PPB caused by both metalaxyl‐sensitive and metalaxyl‐resistant isolates. The data of this study also proved that there was obviously no cross‐resistance between metalaxyl and the other tested fungicides. Therefore, these fungicides should be good alternatives to metalaxyl for the control of PPB and management of metalaxyl resistance.     

Integrating cultural control methods for tomato late blight (Phytophthora infestans) in Uganda

Cultural control measures against tomato late blight (Phytophthora infestans) were evaluated in six field experiments over 3 years in Uganda. Each experiment included sanitation (removal of diseased plant tissues), fungicide (mancozeb) application, and an untreated control, as standard treatments. Late blight incidence and severity were greatly reduced by sanitation, without reducing the number of healthy leaves; however, tomato growth and production were adversely affected. Fungicide treated plants retained the highest numbers of flowers and attached fruits and gave the highest yields. Three cultural practices were evaluated in repeated experiments for their effectiveness in alleviating the adverse effects of sanitation. Tomatoes grown within plastic shelters early in the production cycle were taller, and had more healthy leaves than those grown late. The numbers of diseased leaves and disease severity were equally low in sanitation alone and plastic shelter/with sanitation treatments. Flower and fruit production were significantly higher when tomatoes were grown under early shelters with sanitation than with sanitation alone. Planting density was increased without significant effects on late blight and tomato growth and production. Intercropping tomato with soybean (Glycine max) or sesame (Sesamum indicum), with sanitation, limited late blight development, but taller intercrops suppressed tomato growth and production. Integrated treatments (combining plastic shelters, a sesame intercrop and high tomato planting density) were evaluated, with and without sanitation, against the fungicide mancozeb. The mean numbers of healthy leaves in the integrated treatments were not significantly less than with fungicide treatment. Late blight incidence and severity were higher in the integrated plots without than with sanitation. The numbers of flowers and attached fruits were not significantly less in integrated treatments than in fungicide treated plots, but tomato yield was highest with fungicide treatment.     

The effect of fungicides, irrigation and plant density on the development of Peronospora sparsa, the cause of downy mildew in rose and blackberry

Downy mildew of rose (Rosa spp.) and blackberry (Rubus fructicosus), caused by Peronospora sparsa, can cause significant losses in production. In a series of experiments, fungicides with different modes of action to the commonly used phenylamide‐based products were examined for efficacy on both crops. Cymoxanil + mancozeb + oxadixyl and fluazinam gave good downy mildew control on both rose and blackberry. On outdoor, container‐grown rose, high volume sprays of fosetyl‐aluminium were also effective, but on young micropropagated blackberry plants, application as a drench treatment was better than as a spray. Good control was also achieved on blackberry with chlorothalonil and with metalaxyl in formulation with either thiram or mancozeb. There was no evidence of loss of control with phenylamide fungicides due to resistance. Irrigation regimes were also examined on blackberry and sub‐irrigation of plants in propagation on a sand bed led to significantly lower disease levels than those found where plants had been irrigated by overhead sprinklers. Reducing the density of container‐grown rose plants had a measurable effect on the progress of downy mildew, though this was small and temporary when compared with the effect of fungicide treatment. Fungicide programmes which gave season‐long control of rose downy mildew resulted in significantly improved extension growth.     

Management of the late blight (Phytophthora infestans) disease of potato in the southern hills of India

Late blight of potato is considered to be the most devastating problem causing severe yield losses in potato worldwide. Among the different management strategies, the use of resistant cultivars is the most viable option, but the non‐availability of enough quantity of quality seed materials of resistant cultivars forces the farmers to grow susceptible cultivars with proper fungicide scheduling. Therefore, in the present study, chemical control using fungicide has been attempted with newer molecules in the susceptible cultivar along with a resistant cultivar as a positive control. All the tested fungicides were found safe, and no phytotoxicity was observed with any chemical at the applied rate. In resistant cultivar, no late blight was appeared in both the years, whereas maximum AUDPC was observed in the untreated control (276.3) and minimum (41.7) in mancozeb‐cymoxanil + mancozeb based scheduling which was found on par with chlorothalonil‐famoxadone + cymoxanil (51.3) and chlorothalonil‐ametoctradin + dimethomorph (53.5) based scheduling. Among the treatments, resistant cultivar, Kufri Girdhari followed by chlorothalonil‐ametoctradin + dimethomorph and mancozeb‐cymoxanil + mancozeb based fungicidal scheduling were proven as the best treatments for both the crop seasons resulting in the highest yield parameters. The disease severity showed a strong negative correlation with the tuber yield of potatoes in both the years. Based on overall observations including BC ratio, it can be concluded that, wherever seed material of resistant cultivar is available farmers should grow the same or else with susceptible cultivars the fungicidal scheduling based on mancozeb‐cymoxanil + mancozeb or chlorothalonil‐ametoctradin + dimethomorph can be followed to obtain the maximum returns with effective management of late blight at the southern hills of India.     

The efficacy of spraying programmes and treatment methods against downy mildew in two grapevine cultivars in Kosovo

The present study aimed to assess the efficacy of applied programmes and treatment methods against downy mildew (Plasmopara viticola). Two vine cultivars (Frankovka and Game) were tested in an experimental field (in Rahovec, Kosovo) for a two-year period (2010–2011). The fungicides used were Quadris (azoxystrobin), Antracol EP-70 (propineb), Dithan M-45 (mancozeb), Ridomil (metalaxyl), Curzate (cymoxanil), Bordeaux mixture (calcium hydroxide and copper sulphate) and Mikal (fosetyl-aluminium) applied three treatment methods. The evaluation of the disease severity was performed using the McKinney index. In both tested cultivars and in both treatment years (2010 and 2011), all of the programmes tested significantly reduced the severity of grape downy mildew compared to the control. The highest efficacy against grape downy mildew was achieved with the combined use of Ridomil and Dithan (programme 3); this treatment was more than 80% effective in both cultivars and in both years.     

In vitro Selection of Strains of Phytophthora cactorum Resistant to Metalaxyl

Isolates of Phytophthora cactorum resistant to the systemic fungicide metalaxyl were obtained by exposing them to sequentially increased concentrations of metalaxyl. A linear relationship was observed between the concentrations of metalaxyl and percentage inhibition of mycelial growth of P. cactorum. The stability of metalaxyl-resistant isolates 150R and 250R was confirmed after six serial transfers on corn meal agar without fungicide. The in vitro metalaxyl-resistant isolate (Ph10) was less aggressive on apple rootstocks compared with the Ph07 isolated from metalaxyl-treated trees and the Ph03 isolated from untreated trees. Metalaxyl-resistant and sensitive isolates remained sensitive to the chemically unrelated fungicide fosetyl-Al at high concentration (600 μg/ml), to mancozeb, and to a mixture of metalaxyl + mancozeb. Significant differences in resistance to metalaxyl existed among P. cactorum field isolates.     

Effects of Metalaxyl, Fosetyl-Al, Dimethomorph and Cymoxanil on Phytophthora cactorum of Peach Tree

The activity of metalaxyl, fosetyl-Al, dimethomorph, and cymoxanil against Phytophthora crown rot of peach trees was examined. Application of fosetyl-Al or metalaxyl by painting the trunk (150 g/l) of 3-year-old PR204 trees was inhibitory to growth of the fungus but neither dimethomorph nor cymoxanil were effective. Application of metalaxyl as a soil drench suppressed canker development when the treated trees were subsequently inoculated with Phytophthora cactorum . Fosetyl-Al significantly reduced the growth of fungus compared with cymoxanil, dimethomorph and control but it was not as effective as metalaxyl. Again, dimethomorph and cymoxanil did not influence canker development. Both metalaxyl and fosetyl-Al were active for at least 21 days after applications. Strips of trunk bark were removed from trees, that had been drenched with the tested chemicals 20 days before, and inoculated on the cambium side with P. cactorum . Metalaxyl was the most effective fungicide and fosetyl-Al significantly reduced the development of fungus compared with dimethomorph, cymoxanil and the untreated strips. Colonization of strips treated with dimethomorph was significantly less than untreated strips. In contrast, cymoxanil did not inhibit the growth of fungus. Application of metalaxyl or fosetyl-Al as trunk paint or a soil drench appear to be effective procedures for preventing Phytophthora rot of peach trees.     

Cellular Damage to Phytophthora infestans in Tomato Leaves Treated with Oxadixyl: an Ultrastructural Investigation

Ultrastructural observations showed massive accumulation of electron dense deposits adjacent to plasmalemma and mitochondria and mitochondrial degeneration, which probably leads to cell death, in some hyphae and haustoria of Phytophthora infestans in infected tomato leaves treated with the systemic fungicide oxadixyl (8 μg/ml). However, no changes in the endoplasmic reticulum and number of ribosomes were observed. The cell walls of some hyphae were markedly thickened after oxadixyl treatment. Degenerated haustoria were partly present in intact host cells. Evidently, fungal cell damage by treatment with oxadixyl is not correlated with host cell damage.     

TWO NEW MEMBERS OF THE BLASTOCLADIACEAE. II. MORPHOGENETIC RESPONSES TO O2 AND CO2

The development of Microallomyces dendroideus and Allomyces reticulatus was studied under various mixtures of O₂, CO₂ and N₂. All thalli grown in broth or agar media in air produce abundant resistant sporangia but rarely produce any zoosporangia. In both fungi, the proportion of zoosporangia borne by the thalli increases as the oxygen level decreases. In addition, increased CO₂ strongly stimulates resistant sporangium germination in M. dendroideus. Neither fungus grows in the absence of oxygen; therefore, they seem to be adapted for rapid multiplication in a microaerophilic niche.     

Studies on the metabolism and toxicological detection of the new designer drug N-benzylpiperazine in urine using gas chromatography-mass spectrometry

The metabolism of cyano-oxime fungicide 1-(2-cyano-2-methoxyiminoacetyl)-3-ethylurea (cymoxanil) and analogs was studied on several strains of the fungus Botrytis cinerea owing to their difference in sensitivity towards cymoxanil. Chromatographic analysis of the unextracted culture medium was simpler and more accurate, particularly for ionizable metabolites because it avoids problems associated with extraction. Reversed-phase high-performance liquid chromatography was applied to compare the decrease of cymoxanil and analogs caused by different strains of B. cinerea, by periodic injections of incubated culture medium aliquots, directly on a C4 wide-pore column. Furthermore, a thin-layer chromatographic monitoring on C18 bonded silica gel with ion-pairing allowed the monitoring of the ionizable metabolites for substrates that were demonstrated to decompose most rapidly. These complementary analyses showed that the sensitivity of the highly sensitive strain towards cymoxanil was related to the disappearance of cyano-oximes studied from culture medium, namely to the ability of the strain B. cinerea to metabolize them.     

Biocontrol of tomato late blight with the combination of epiphytic antagonists and rhizobacteria

Control of tomato late blight (LB) in Brazil is heavily based on chemicals. However, reduction in fungicide usage is required in both conventional and organic production systems. Assuming that biological control is an alternative for LB management, 208 epiphytic microorganisms and 23 rhizobacteria (RB) were isolated from conventional and organically grown tomato plants and tested for antagonistic activity against Phytophthora infestans. Based on in vitro inhibition of sporangia germination and detached leaflet bioassays, four EP microorganisms (Aspergillus sp., Cellulomonas flavigena, Candida sp., and Cryptococcus sp.) were selected. These microorganisms were applied either singly or combined on tomato plants treated or not with the RB Bacillus cereus. On control plants, LB progress rate (r), area under disease progress curve, and final disease severity were high. Lowest values of final severity were recorded on plants colonized by B. cereus and treated with C. flavigena, Candida sp. and Cryptococcus sp. There was no reduction on disease severity in plants treated only with RB. Biological control of LB resulted in low values of r and final severity. Integration of biological control with fungicides, cultural practices, and other measures can contribute to manage LB on tomato production systems.     

Biology and Control of Entyloma eryngii (Cda.) de By., the Cause of a Leaf Spot Disease on the Blue Thistle (Eryngium alpinum L.)

The blue thistle (Eryngium alpinum L.), an indigenous perennial flower of the alps, is grown in mountainous regions of south-eastern Switzerland. The commercial value of the fresh cut flowers can be seriously reduced due to a leaf spot disease caused by the smut fungus Entyloma eryngii (Cda.) de By. The fungus produces thick walled chlamydospores and two types of conidia. The disease appears to be favoured by cool-rainy weather and a dense foilage resulting in long periods of leaf wetness. Control of the disease can be achieved with the protective fungicide mancozeb or even more efficiently with the systemic ergosterol-biosynthesis-inhibiting fungicides cyproconazole and difenoconazole.     

Resistance of rin and nor tomato mutants to postharvest Rhizopus infection

Fruits of the two non-ripening mutants of tomato, rin and especially nor, were markedly more resistant to Rhizopus stolonifer infection than the normal Rutgers fruit. Following artificial inoculations by contact with a diseased normal tomato covered with mycelium and sporangia, no infection of unwounded nor fruit occurred at its mature-green stage. At the mature stage the resistance of nor mutant fruit was manifested by a prolongation of the incubation period of the disease as well as by a markedly reduced incidence of rotted fruits. Chilling injury of fruit, prior to spore inoculation, was found to be a good means for indicating the relative resistance of the mutants as compared with the normal tomato. The relationship between the resistance of the mutant tomatoes to Rhizopus infection and their response to induced peel damage as a result of the contact or the chilling procedure, led to the assumption that fruit resistance is associated with the inability of the fungus to penetrate the periderm, rather than with fungal development within the fruit.     

Effects of Selected Fungicides and the Timing of Fungicide Application on Beauveria bassiana-Induced Mortality of the Colorado Potato Beetle (Coleoptera: Chrysomelidae)

Four fungicides used for controlling foliar diseases of potato (Solanum tuberosum) were evaluated under field and laboratory conditions for their effects on the infectivity and sporulation of Beauveria bassiana when used as a control for the Colorado potato beetle, Leptinotarsa decemlineata (CPB). We investigated the direct effects of fungicides on B. bassiana-induced CPB mortality and the effect of time between fungicide and B. bassiana application. Effects of fungicide on conidial survival in the soil and on foliage were examined in the field. Significantly more larval mortality was observed when larvae were sprayed with B. bassiana than with the water control. Fungicide had no significant effect on larval mortality in the field. In the laboratory, survival of larvae was significantly lower among larvae fed fungicide-treated foliage. B. bassiana-induced mortality in the laboratory was observed only when larvae were fed foliage treated with copper hydroxide or water. Larvae fed mancozeb- or chlorothalonil-treated foliage experienced high mortality regardless of B. bassiana treatment. While there was no significant effect of fungicide on B. bassiana sporulation on cadavers in the field, a pattern emerged that indicated higher proportions of cadavers producing conidia in plots sprayed with water or copper hydroxide than in plots sprayed with chlorothalonil or mancozeb. Survival of B. bassiana conidia in the soil and on foliage was significantly greater in plots treated with copper hydroxide or water than in plots treated with mancozeb or chlorothalonil. Fungicides such as copper hydroxide may be less deleterious to the fungus than mancozeb and chlorothalonil.     

Inhibitory Effect of Paenibacillus polymyxa GBR-462 on Phytophthora capsici Causing Phytophthora Blight in Chili Pepper

In the present work 25 strains of Paenibacillus polymyxa isolated from rotted ginseng roots were screened for their antimicrobial activity against Phytophthora capsici in vitro . Based on antimicrobial activity, 15 strains categorized as strongly antimicrobial, among them GBR-462 was found as the most active, and five strains each as weekly antimicrobial and no antimicrobial. Antimicrobial activity was influenced by the initial inoculum density, as strains of P. polymyxa with a strong antimicrobial activity (including P. polymyxa GBR-462) showed the antimicrobial activity against P. capsici and could form biofilm only when they were applied at the higher initial inoculums, 10⁸ cfu/ml. No inhibitory effect was noted on the mycelial growth and zoospore germination of the pathogen when applied at the lower inoculum density of 10⁶ cfu/ml of P. polymyxa GBR-462. However, sporangium formation and zoospore release was significantly inhibited at the lower inoculum density. Also light and electron microscopy revealed the structures of sporangia aberrant with no or few healthy nuclei, indicating sporangium and zoospore formation inhibited at the lower inoculum density. Application of P. polymyxa GBR-462 into potted soil suppressed disease progression as well as disease severity; disease severity was reduced by 30% as compared to untreated pots, suggesting P. polymyxa GBR-462 could be a potential biocontrol agent against Phytopthora capsici .     

In vitro and in vivo bio-assay of phytobiocidal effect of plant extracts on Alternaria solani causing agent of early blight disease in tomato

The efficacy of six locally available plants extract was evaluated for their phytobiocidal effect on Alternaria solani the causal agent of early blight (EB) disease of tomato and was compared with commercial fungicide mancozeb under in vitro and in vivo conditions. Under in vitro conditions, Eucalyptus globus and Datura alba were found to be ineffective, while the remaining plants extract (Allium sativum, Curcuma longa, Melia azedarach, Zingiber officinale) significantly reduced A. solani growth on PDA. Increasing concentrations of A. sativum (0–25%) were found negatively correlated with growth of A. solani on PDA. Among the tested plants, A. sativum extract at 20% concentration was found most effective against A. solani witnessed by both in vitro and in vivo experiments result. It reduced EB disease up to 75.11% over positive control. Similarly, among the different plants extract, maximum plant height (76.25 cm), fruit size (57.50 cm³) and yield (511.30 g) were observed in A. sativum sprayed treatments (20%), beside mancozeb and negative control. Upon phytochemical analysis of these extracts, flavonoids, alkaloids, saponins, tannins, terpenoids, glycosides and steroids were detected. Present study showed that 20% concentration of garlic extract has the potential to reduce EB disease severity, while having no noticeable phytotoxicity.     

A spectrophotometric approach for determining sporangium and zoospore viability of Plasmopara viticola

A spectrophotometric method for determining the viability of sporangia and zoospores of the oomycete Plasmopara viticola (causal agent of grapevine downy mildew) is described and evaluated to overcome the limitations of currently available methods for assessing propagule viability. Sporangia produced on leaf discs in the laboratory were harvested at different days after the initiation of sporulation (DAS) to yield differences in sporangium viability. Sporangia were suspended in sterile water, the suspensions were placed in a cuvette, and sporangium germination was monitored in a spectrophotometer (λ = 600 nm) at 2- to 3-min intervals for 5 hr. Absorbance started to increase after sporangia were suspended in water for ~30–60 min followed by major peak(s) for younger sporangia (1–3 DAS), whereas low to no increase in absorbance was observed for senescent sporangia (>7 DAS). Microscopic observation confirmed that the increase in absorbance corresponded to the release and active swimming of zoospores, whereas absorbance decreased when zoospores encysted and settled. A positive correlation (r = .839, p = .0365) was observed when the time to the initial increase in absorbance was plotted against the age of sporangia. The time to the absorbance peak (marking the time of maximum zoospore movement) was shortest for immature sporangia (0 DAS), longest for young sporangia (2 DAS) and decreased for mature and senescent sporangia. A similar pattern was observed for the standardized area under the absorbance curve (indicating the overall quantity of zoospores released), for which values were lowest for immature and senescent sporangia, highest for young sporangia and intermediate for mature sporangia. Consistent patterns obtained across two independent experiments suggest that the method is reproducible and may be further developed for other zoospore-releasing pathogens.