Fungicide resistance in cucurbit downy mildew – methodological, biological and population aspects

Cucurbit downy mildew, caused by Pseudoperonospora cubensis, is among the most devastating diseases of cucurbitaceous plants. In spite of improved cultural practices and breeding for resistant cultivars, chemical control is still a very important tool to manage the disease. During the last several decades, many fungicides from various chemical classes have been developed. The occurrence of strains of P. cubensis resistant/tolerant to some fungicides encouraged research of this phenomenon. The first part of this article summarises the many different methodological approaches such as field trials, in vitro testing on active plant tissues or molecular diagnoses developed for the detection of resistant/tolerant strains of P. cubensis, as well as methods to collect and maintain pathogen isolates. The second part outlines the commonly used fungicides to control P. cubensis and their features like systemicity, biological and biochemical mode of action and translocation behaviour within plants. The last part deals with geographical aspects such as first appearance of resistance problems, distribution of resistance, temporal development of resistance under selection pressure by a fungicide, fitness of resistant subpopulations in competition with sensitive ones in the absence of a fungicide, as well as genetic and molecular sources of resistance.     

Utilization of Caffeic Acid Phenethyl Ester to Control Alternaria alternata Rot in Tomato (Lycopersicon esculentum Mill.) Fruit

Chemical fungicides that are related with resistant strains develop negative effects on human health and environment. Propolis is a resinous substance collected by bees with positive effects on human health and inhibitory activity against Alternaria alternata. Caffeic acid phenethyl ester (CAPE) is a component of the propolis. The objective of this experiment was to test the effect of CAPE on fungi infecting tomato fruit using as a model the pathosystem A. alternata‐tomato. CAPE was chemically synthesized in our laboratory and analysed with nuclear magnetic resonance spectroscopy. Different concentrations (0, 16, 32, 48, 64, 80, 90 and 100 μm ) of CAPE were tested on A. alternata growing in vitro. For the in vivo experiment, red ripe tomato fruit was inoculated with A. alternata and untreated or treated with 1, 50 and 100 μm of CAPE. After that, the fruit was stored at 25°C for up to 20 days. Colony size (CS) was recorded in vitro. In tomato fruits, the severity of infection (SI), respiration rate (RR), ethylene production (EP), pH, total soluble solids (TSS), weight loss (WL) and titratable acidity (TA) were evaluated during the storage time. CAPE melting point and spectral data probed to be the right molecule. In vitro, 64 and 100 μm of CAPE reduced CS by 30%. In vivo, 50 and 100 μm of CAPE reduced SI higher than the fungicide Captan^® with no effects on RR, EP, WL, pH, TSS and TA. It was concluded that CAPE controls A. alternata infection better than a commercial fungicide without negative effects on tomato fruit ripening and fruit quality.     

Damping-off of Some Cucurbitaceous Crops in Saudi Arabia with Reference to Control Methods

Isolation of seed borne fungi from sweet melon (najed and red Queen varieties) and vegetable marrow (squash) using PDA, Plain agar media and blotters revealed the presence of Alternaria alternata (Fr.) Keissler, Fusarium semitectum var,majus from Najed melon, A. alternata (Fr.) Keissler, F. sambucinum Fuckel from Red Queen melon and A. chlamydospora Mouchacca, Cephalosporium sp., and F. oxysporum schlecht from squash. Pathogenicity tests showed, that all these fungi were highly pathogenic on their respective hosts. The optimum temperature for its growth ranged from 25–30°C and the optimum pH was 6.0. In pot trials, seed dressing with Banrot, Bavistin and Topsin-M at the rate of 4 g/kg seed was superior in controlling the damping-off of melon and squash. These fungicides were effective in inhibiting mycelial growth, spore germination and development of the isolated fungi. Hot water treatment at 55 C/20 min or solar heating (av. 37°C) for 90 min were next to fungicides.     

Resistance reactions of Cucumis melo to inoculation with Pseudoperonospora cubensis

Reactions of several cultivars and breeding lines of melons to artificial inoculation at the seedling stage with a local isolate of Pseudoperonospora cubensis were compared under growth chamber and field conditions. Four different reactions were distinguishable: highly susceptible, susceptible, partially resistant and resistant. The differences in the reactions were more clear-cut under growth chamber conditions. Selection for resistance could be carried out at the seedling stage using sporangia production and lesion size, shape, colour and number as criteria. Under growth chamber conditions, plants of resistant lines had minimal sporangia production and circular or angular lesions 1–2 cm in diameter which became necrotic within 5 days after inoculation.     

Towards better use of Indonesian peatlands with paludiculture and low-drainage food crops

The current drainage-based peatland management systems in Indonesia result in high fire risks, soil subsidence and CO₂ emissions. This study aims to assess different alternatives of peatland crops in order to help prevent further degradation of peatlands in Indonesia. We focus on tropical peatland crops that provide food and that are of particular interest to smallholders. We compare various peatland food crops that are commonly grown with no drainage (paludiculture) or drainage below 50 cm in our study area, Central Kalimantan, Indonesia in terms of sustainability, profitability, scalability of the market and acceptability to farmers. Our results show that sago (Metroxylon sagu), banana (Musa paradisiaca) and pineapple (Ananas comosus) followed by water spinach/kangkong (Ipomoea aquatica), kelakai/edible fern (Stenochlaena palustris), illipe nut/tengkawang (Shorea spp.), dragon fruit (Hylocereus undatus), mangosteen (Garcinia mangostana) and sweet melon/melon (Cucumis melo) are the best options based on the aggregated scores for these criteria (but precaution should be taken when planting crops that require low drainage). Sago palm and illipe nut have the highest scores for both sustainability and scalability of market, whereas banana, pineapple and sweet melon have the highest scores in term of the scalability of market and acceptability to farmers. We also address key opportunities and bottlenecks for the development of paludiculture food crops and present recommendations for the implementation of paludiculture in Indonesian peatlands.

     

Selection of a Suitable Medium to Determine Sensitivity of Monilinia fructicola Mycelium to SDHI Fungicides

Succinate dehydrogenase inhibitor (SDHI) fungicides are important tools to control preharvest and postharvest brown rot of stone fruits. In this study we determined the mycelial growth rate of Monilinia fructicola isolates on various media and picked the three that allowed the fastest growth to assess the sensitivity of mycelium to SDHI fungicides boscalid, fluopyram, and penthiopyrad. Minimal medium (MM) supported mycelial growth the best and yielded lowest EC₅₀ values for three SDHI fungicides. EC₅₀ values corresponded with disease incidence data obtained from detached fruit assays. Penthiopyrad had significantly greater intrinsic activity in vitro compared to fluopyram at the α = 0.05 level and compared to boscalid at the α = 0.1 level. However, detached fruit assays revealed that this ‘advantage’ did not carry over in vivo. In conclusion, MM appears to be the best medium currently available to assess the sensitivity of M. fructicola mycelium in vitro.     

Volatile Components of Roasted Chufa-tubers

The desmutagenic activities of some vegetable and fruit juices to the mutagenicity of 2-tert-butyl-p-quinone (t-BQ) were assayed by the Ames test using Salmonella typhimurium TA 100. t-BQ is a strong mutagen and is produced from butylated hydroxyanisole when treated with nitrite under acidic conditions. The juices of sweet pepper, lemon, tomato, orange, strawberry, melon, and kiwi fruit reduced the mutagenicity of t-BQ, the desmutagenic activities being proportionally related to the content of the sulfhydryl group contained in these juices. The authentic sulfhydryl compounds, glutathione, pantetheine and dithiothreitol, were then tested for their desmutagenicity against t-BQ by the induced-mutation frequency method, and they were proved responsible for the desmutagenicity. Glutathione reduced half of the t-BQ to 2-tert-butyl-hydroquinone during the desmutagenic reaction, the other half being suggested to have been a conjugated compound between glutathione and t-BQ.     

Melon Fruits: Genetic Diversity,Physiology, and Biotechnology Features

Among Cucurbitaceae, Cucumis melo is one of the most important cultivated cucurbits. They are grown primarily for their fruit, which generally have a sweet aromatic flavor, with great diversity and size (50 g to 15 kg), flesh color (orange, green, white, and pink), rind color (green, yellow, white, orange, red, and gray), form (round, flat, and elongated), and dimension (4 to 200 cm). C. melo can be broken down into seven distinct types based on the previously discussed variations in the species. The melon fruits can be either climacteric or nonclimacteric, and as such, fruit can adhere to the stem or have an abscission layer where they will fall from the plant naturally at maturity. Traditional plant breeding of melons has been done for 100 years wherein plants were primarily developed as open-pollinated cultivars. More recently, in the past 30 years, melon improvement has been done by more traditional hybridization techniques. An improvement in germplasm is relatively slow and is limited by a restricted gene pool. Strong sexual incompatibility at the interspecific and intergeneric levels has restricted rapid development of new cultivars with high levels of disease resistance, insect resistance, flavor, and sweetness. In order to increase the rate and diversity of new traits in melon it would be advantageous to introduce new genes needed to enhance both melon productivity and melon fruit quality. This requires plant tissue and plant transformation techniques to introduce new or foreign genes into C. melo germplasm. In order to achieve a successful commercial application from biotechnology, a competent plant regeneration system of in vitro cultures for melon is required. More than 40 in vitro melon regeneration programs have been reported; however, regeneration of the various melon types has been highly variable and in some cases impossible. The reasons for this are still unknown, but this plays a heavy negative role on trying to use plant transformation technology to improve melon germplasm. In vitro manipulation of melon is difficult; genotypic responses to the culture method (i.e., organogenesis, somatic embryogenesis, etc.) as well as conditions for environmental and hormonal requirements for plant growth and regeneration continue to be poorly understood for developing simple in vitro procedures to culture and transform all C. melo genotypes. In many cases, this has to be done on an individual line basis. The present paper describes the various research findings related to successful approaches to plant regeneration and transgenic transformation of C. melo. It also describes potential improvement of melon to improve fruit quality characteristics and postharvest handling. Despite more than 140 transgenic melon field trials in the United States in 1996, there are still no commercial transgenic melon cultivars on the market. This may be a combination of technical or performance factors, intellectual property rights concerns, and, most likely, a lack of public acceptance. Regardless, the future for improvement of melon germplasm is bright when considering the knowledge base for both techniques and gene pools potentially useable for melon improvement.     

MORPHOLOGY AND LIFE CYCLE OF A NEW CHYTRID WITH AERIAL SPORANGIA

An investigation of the life cycle of Caulochytrium gloeosporii Voos and Olive, a parasite of Gloeosporium, has confirmed the existence of an alternation of generations. Zoospores from sessile sporangia may develop vegetatively into similar sporangia or they may function as isogametes, fusing in pairs to produce cysts (zygotes) that give rise to slender-stalked aerial sporangia. Meiosis is believed to occur in the aerial sporangium, which at germination liberates eight asexual zoospores. The species is homothallic.     

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.     

Effect of pre-harvest fungicides on the spoilage of soft fruit after harvest

In the 1972 season, Elvaron and Benlate reduced the count of Botrytis cinerea, Cladosporium spp., Penicillium spp. and Aureobasidiutn pullulans on strawberry and raspberry fruits during the first 2 wk of the season, but not subsequently. No such reduction was recorded for Hainesia lythri (strawberries only), Phoma state of Didymella applanata (raspberries only), Mucor spp., and Rhizopus spp. Similar results were obtained in 1973. In the first half of either season, both fungicides decreased the rate of fungal spoilage of stored fruit (except for Benlate in the 1973 season), although in the latter part of the season, there was little difference in the incidence of fungal spoilage between sprayed and unsprayed fruit. Both fungicides, however, reduced the development of B. cinerea (and Cladosporium spp. on raspberries) on stored fruit irrespective of season or harvest date except for Benlate in the 1973 season where Benlate-resistant strains of the fungi developed on the fruit. The failure of either fungicide to reduce fungal spoilage in the latter part of the harvesting season was due to the development of Phycomycetes, particularly Mucor mucedo and to a lesser extent Rhizopus stolonifer and R. sexualis, except where Benlate-resistant strains of B. cinerea and Cladosporium spp. were present.     

Distribution,Host Range and Disease Severity of Pseudoperonospora cubensis on Cucurbits in the Czech Republic

Cucurbit downy mildew, caused by Pseudoperonospora cubensis, is a major cucumber disease in the Czech Republic. Disease prevalence, host range and disease severity were evaluated from 2001 to 2009. The geographical distribution of P. cubensis was assessed on ca 80–100 locations per year in two main regions of the Czech Republic (central and southern Moravia, and eastern, northern and central Bohemia). Infection by P. cubensis was observed primarily on cucumber (Cucumis sativus) but only on the leaves. During the study, disease prevalence ranged from 66 to 100%. The majority of C. sativus crops were heavily infected at the end of the growing season (second half of August). Generally, P. cubensis was present at high or very high disease severity. The loss of foliage results in the reduction in the quality and quantity of marketable yield of fruit. Pseudoperonospora cubensis was widespread across the whole area of the Czech Republic studied. Very rarely, infection was recorded in muskmelon (Cucumis melo) and Cucurbita moschata. Of other pathogens, the most frequently recorded was the cucurbit powdery mildew (Golovinomyces cichoracearum and Podosphaera xanthii).     

Effect of fungicides on the mycelial growth of soft fruit spoilage fungi

The in vitro activity of a range of fungicides was tested on strains of Botrytis cinerea, Mucor mucedo, Rhizopus stolonifer, and R. sexualis. Di-chlofluanid and dichloran were more active than the other fungicides against all of the strains of B. cinerea, while dichloran, dichlorophen and the dithiocarbamate fungicides were the most active against the Rhizopus species. Only dichlorophen and thiram markedly inhibited the growth of M. mucedo.     

甜瓜乙烯应答因子基因在果实发育成熟过程中的表达特性

根据已报道的甜瓜CMe-ERF1和CMe-ERF2基因cDNA序列设计合成特异性引物,应用RT-PCR技术从甜瓜品种‘河套蜜瓜’成熟果实中克隆得到CMe-ERF1和CMe-ERF2基因cDNA全长编码区序列,分别为498bp和822bp.序列比对分析表明,得到的cDNA序列与已报道的Andes甜瓜相应基因的cDNA序列完全一致.果实不同发育时期实时定量PCR检测结果表明,CMe-ERF1、CMe-ERF2基因表达与甜瓜果实成熟及乙烯生成量显著相关,表明该基因可能对果实成熟起重要作用.     

Physical and biological compatibility of diafenthiuron with micro/macro nutrients fungicides and biocontrol agents used in cardamom

In the context of complex field problems, compatibility of an efficacious insecticide with other agrochemicals normally used in the field is essential. In this view diafenthiuron, a novel insecticide which inhibits ATP synthesis, used widely for pest management in cardamom, was tested for its compatibility with agrochemicals viz., fungicides and nutrients normally used in the crop. The results revealed that all the chemicals tested were physically and biologically compatible with diafenthiuron by means of physical stability and phytotoxicity ratings in the field. But the bioefficacy study on Conogethes punctiferalis in the laboratory and bioefficacy studies in the field against Sciothrips cardamomi reveal that diafenthiuron is incompatible and should not be sprayed along with fungicides like mancozeb and copper oxychloride. Another study on the compatibility of diafenthiuron with antagonistic microorganisms of plant pathogens viz., Trichoderma viride and Pseudomonas fluorescens revealed that diafenthiuron had some inhibitory effect on the mycelial growth of T. viride. Diafenthiuron did not affect the growth of P. fluorescens and thus can be used simultaneously for the control of insect pests and seed- and soil-borne diseases.     

Catabolism of l–methionine in the formation of sulfur and other volatiles in melon (Cucumis melo L.) fruit

Sulfur‐containing aroma volatiles are important contributors to the distinctive aroma of melon and other fruits. Melon cultivars and accessions differ in the content of sulfur‐containing and other volatiles. l –methionine has been postulated to serve as a precursor of these volatiles. Incubation of melon fruit cubes with ¹³C‐ and ²H‐labeled l –methionine revealed two distinct catabolic routes into volatiles. One route apparently involves the action of an l ‐methionine aminotransferase and preserves the main carbon skeleton of l ‐methionine. The second route apparently involves the action of an l ‐methionine‐γ–lyase activity, releasing methanethiol, a backbone for formation of thiol‐derived aroma volatiles. Exogenous l ‐methionine also generated non‐sulfur volatiles by further metabolism of α–ketobutyrate, a product of l ‐methionine‐γ–lyase activity. α–Ketobutyrate was further metabolized into l –isoleucine and other important melon volatiles, including non‐sulfur branched and straight‐chain esters. Cell‐free extracts derived from ripe melon fruit exhibited l ‐methionine‐γ–lyase enzymatic activity. A melon gene (CmMGL) ectopically expressed in Escherichia coli, was shown to encode a protein possessing l ‐methionine‐γ–lyase enzymatic activity. Expression of CmMGL was relatively low in early stages of melon fruit development, but increased in the flesh of ripe fruits, depending on the cultivar tested. Moreover, the levels of expression of CmMGL in recombinant inbred lines co‐segregated with the levels of sulfur‐containing aroma volatiles enriched with +1 m/z unit and postulated to be produced via this route. Our results indicate that l ‐methionine is a precursor of both sulfur and non‐sulfur aroma volatiles in melon fruit.     

Variation of Fungicide Resistance in Czech Populations of Pseudoperonospora cubensis

During the growing seasons between the years 2001 and 2004, 98 isolates of Pseudoperonospora cubensis from nine regions of Czech Republic were collected and screened for tolerance/resistance to the three frequently used fungicides (propamocarb, fosetyl‐Al, metalaxyl). Fungicides were tested in five different concentrations, using a floating disc bioassay. Fungicide effectiveness varied considerably. Propamocarb appeared most effective and all the isolates collected in the years 2001–2003 were found sensitive to all tested concentrations [607–9712 μg active ingredient (a.i.)/ml]. In 2004, some strains with increased resistance to propamocarb were detected. These strains were characterized by tolerance at the lowest concentrations (607 μg a.i./ml, eventually on 1214 μg a.i./ml); however, they were controlled by 2428 μg a.i./ml. Fosetyl‐Al was effective at the recommended concentration of 1600 μg a.i./ml against all isolates. However, the occurrence of isolates (collected in 2001) which sporulated at low concentrations (400 and 800 μg a.i./ml) indicated that the selection for tolerance occurs in the pathogen population. Nevertheless, this phenomenon was not confirmed with the P. cubensis isolates collected between the years 2002 and 2004. Metalaxyl was found ineffective, because 97% of the isolates showed the resistance to the recommended concentration (200 μg a.i./ml), and the other 3% of isolates expressed tolerant response. The majority of the isolates showed profuse and/or limited sporulation at higher concentrations (400 and 800 μg a.i./ml). A substantial shift to highly metalaxyl resistant strains was evident in the Czech P. cubensis populations during 2001–2004.     

Morphology and life history of Scytosiphon canaliculatus comb. nov. (Scytosiphonales,Phaeophyceae) from Japan*

Morphology of field material and life history in culture were studied in Scytosiphon canaliculatus (Setchell et Gardner) comb. nov. from northern Japan. Erect gametophytes of S. canaliculatus are cylindrical, tubular, up to 7 mm wide and 40 cm long, and without regular constrictions. S. canaliculatus has pronounced anisogamy and ascocysts accompanied with plurilocular gametangia. The life history of S. canaliculatus showed an alternation between erect gametophytes and crustose prostrate sporophytes bearing unilocular sporangia. Since field sporophytes of S. canaliculatus were found to be identical with Hapterophycus canaliculatus Setchell et Gardner (Ralfsiaceae, Phaeophyceae), it is proposed to transfer H. canaliculatus to the genus Scytosiphon. In the field, gametophytes with plurilocular gametangia appeared in spring and disappeared in summer. Sporophytes with unilocular sporangia were collected in late autumn and winter. Unilocular sporangia were produced at 15°C in short-day culture conditions and unispores developed into erect gametophytes at 5–15°C. It is suggested that the seasonal Occurrence of gametophytes in the field is due to the seasonal formation of unilocular sporangia, which is regulated by temperature and photoperiod.     

Effect of carbendazim and mancozeb combinationon Alternaria leaf blight and seed yield in sunflower (Helianthus annus L.)

Abstract

Leaf blight caused by Alternaria helianthi (Hansf.) Tubaki & Nishihara, is the major disease of sunflower affecting the successful cultivation across India. Five individual fungicides and two combination fungicides were evaluated against this pathogen in laboratory and in field experiments. Among them, the combination of carbendazim + mancozeb completely (100%) inhibited the mycelial growth of A. helianthi, irrespective of the concentrations tested followed by carbendazim alone and metalaxyl + mancozeb under in vitro condition. In field conditions, the combination of carbendazim + mancozeb was found to be highly effective in reducing the leaf blight disease of sunflower in all the three experiments as compared to other fungicides and unsprayed control. The reduction of Alternaria leaf blight was also directly associated with an increase in seed yield. The economics of the fungicides spray has been worked out and the benefit cost ratio for the combination of carbendazim + mancozeb at 2.0 g/l was 7.1 as compared to unsprayed control. The overall analysis of the results revealed that the combination of carbendazim + mancozeb at 2.0 g/l can be used for the management of foliar diseases such as Alternaria leaf spot/blight in agricultural crops.