Assessing the Sensitivity of Plasmopara halstedii Isolates to Mefenoxam through Host Responses.

Downy mildew caused by Plasmopara halstedii is responsible for significant economic losses in cultivated sunflowers. Field isolates of sunflower downy mildew resistant to mefenoxam, a previously effective active ingredient against the pathogen, have been found across Europe. The main goal of this study was to assess the sensitivity of P. halstedii isolates to mefenoxam through host responses to infection, such as symptoms measured by disease severity and growth reduction, and host tissue reactions, such as hypersensitive reaction and necrosis of invaded cells. Sunflower seeds were treated with Apron XL 350 FS at the European registered rate (3 mg/kg seeds). Seedlings were inoculated using the soil drench method with eight Hungarian P. halstedii isolates. Disease rates and plant heights were measured twice. Histological examinations of cross-sections of sunflower hypocotyls were performed using a fluorescence microscope. In our study, cluster analyses of sunflowers based on macroscopic and microscopic variables showed differentiation of groups of mefenoxam-treated sunflowers inoculated with different P. halstedii isolates. We first revealed a clear difference in host responses of mefenoxam-treated susceptible sunflowers. In addition, examining tissue reactions (e.g., hypersensitive reaction, necrosis) seems more accurate to estimate the sensitivity of P. halstedii isolates to mefenoxam than macroscopic symptoms.

New and High Virulent Pathotypes of Sunflower Downy Mildew (Plasmopara halstedii) in Seven Countries in Europe.

Downy mildew of sunflower, caused by Plasmopara halstedii (Farl.) Berl. et de Toni, is a relevant disease of this crop. High virulent pathotypes have been identified in several countries, while there are few data on the spread of P. halstedii pathotypes in some important sunflower-growing areas of Europe. The goal of this study was to give up-to-date information on the pathotype structure of P. halstedii in Hungary and provide some actual data on the virulence phenotype of the pathogen for six European countries. Infected leaves of different sunflower hybrids and volunteers were collected in seven countries (Hungary, Bulgaria, Serbia, Turkey, Greece, Romania, and Italy) between 2012 and 2019. A universally accepted nomenclature was used with a standardized set of sunflower differential lines for pathotype characterization of isolates. The virulence pattern of the isolates was determined by a three-digit code (coded virulence formula, CVF). A total of 109 P. halstedii isolates were characterized. As a result of our survey, 18 new P. halstedii pathotypes were identified in Europe. Two out of the eighteen pathotypes were detected from the Asian part of Turkey. The detailed distribution of pathotypes in Hungary is also discussed.

First Report on the Occurrence of an Aggressive Pathotype, 734, of Plasmopara halstedii Causing Sunflower Downy Mildew in Hungary.

Downy mildew of sunflower (Helianthus annuus L.) is caused by Plasmopara halstedii (Farl.) Berl. et de Toni, leading to significant losses in crop production worldwide. The number of new and more aggressive pathotypes has increased rapidly over the last 10 years in Europe (Virányi et al. 2015, Bán et al. 2018), therefore, constantly monitoring the distribution of races is an important task. As part of regular surveys in June 2019, severe downy mildew was identified in some regions, appearing as chlorotic lesions along the veins of the adaxial side and white sporulation on the abaxial side of the leaves of severely stunted hybrids containing PI6 and PI7 resistance genes. The identification of the pathogen was performed microscopically based on morphological characteristics (average size of sporangia: 28x20 µm). Disease incidence (the ratio of diseased plants) ranged between 10 and 30% per field in three regions and resulted in moderate yield loss. Isolates (defined as a lesion per leaf) were collected from 4 to 8 infected leaves of each hybrid by region and stored at -70°C. Two, one and one isolates of P. halstedii were selected and characterized from the southeastern (Békés County), northern (Nógrád County) and northeastern (Borsod-Abaúj-Zemplén County) regions of Hungary, respectively. The pathotype of the four isolates was determined using the international standardized nomenclature method reviewed by Trojanová et al. (2017), including nine sunflower differential inbred lines (HA-304, RHA-265, RHA-274, PMI-3, PM-17, 803-1, HAR-4, QHP2 and HA-335). Zoosporangia from frozen sunflower leaves were washed off into bidistilled water and the concentration was adjusted to 3.5x104 sporangia/ml using a hemocytometer. Three-day-old seedlings with a radical of 1 to 1.5 cm long were immersed in the sporangial suspension and kept at 16°C overnight (Cohen and Sackston 1973). Inoculated seedlings were planted into trays containing clear moistened perlite (d = 4 mm) and grown in a growth chamber with a photoperiod of 12 h. The experiment was carried out twice with each isolate using 15 seeds/differential line with two replicates. Bidistilled water was sprayed on the plants 9 days after inoculation, and then trays were covered with a black polyethylene bag and maintained at 19°C overnight to induce sporulation. The first disease assessment was done based on cotyledons bearing white sporulation. Next, a second evaluation was performed 21 days after inoculation assessing stunting of the plants, chlorotic lesions on true leaves and damping-off. All 4 isolates examined caused disease on differential lines HA-304, RHA-265, RHA-274, PMI-3, PM-17 and HA-335, whereas the other lines showed no symptoms and signs of sunflower downy mildew. As a result, it was concluded that the presence of P. halstedii pathotype 734 was confirmed. This pathotype is likely widespread in Hungary as it could be detected from three different regions. Moreover, the possibility that pathotype 734 is present in Hungary has been raised before (Iwebor et al. 2018). This pathotype is already widespread in the USA and Russia and is considered to be highly aggressive, since it was able to infect hybrids with resistance genes PI6 and PI7 (Iwebor et al. 2018, Spring 2019). To our knowledge, this is the first report of pathotype 734 of P. halstedii in Hungary and Central Europe. Continuous monitoring and incorporation of new resistance genes into sunflower hybrids are essential steps in the future to control P. halstedii.

Preliminary Investigation of Effect of Neem-Derived Pesticides on Plasmopara halstedii Pathotype 704 in Sunflower under In Vitro and In Vivo Conditions.

Two neem-derived pesticides were examined under in vitro and in vivo conditions to test their efficacy in controlling Plasmopara halstedii pathotype 704, a causal agent of downy mildew in sunflower. All the tested concentrations of neem leaf extract and the highest concentration of commercial neem product significantly reduced the sporangial germination under in vitro conditions. In in vivo experiment, 3-days old pre-treated seedlings with both concentrations of neem leaf extract and the highest concentration of commercial product showed a significant reduction in the infection indicating possible systemic effect of neem. When the seedlings were treated following the infection with P. halstedii (i.e., post-treatment), only the highest concentrations of neem leaf extract and the commercial product showed a significant reduction in the infection indicating curative effect of neem. Possibilities for the control of P. halstedii with neem-derived pesticides are discussed.

The chemical inducer, BTH (benzothiadiazole) and root colonization by mycorrhizal fungi (Glomus spp.) trigger resistance against white rot (Sclerotinia sclerotiorum) in sunflower.

White rot caused by Sclerotinia sclerotiorum (SS) is one of the most devastating plant diseases of sunflower. Controlling this pathogen by available tools hardly result in acceptable control. The aim of this study was to elucidate the effects of plant resistance inducers, BTH (benzothiadiazole in Bion 50 WG) and arbuscular mycorrhizal fungi (AMF) on disease development of white rot in three sunflower genotypes. Defence responses were characterized by measuring the disease severity and identifying cellular/histological reactions (e.g. autofluorescence) of host plants upon infection. Depending on the host genotype, a single application of inducers reduced disease symptoms. Histological examination of host responses revealed that BTH and/or AMF pre-treatments significantly impeded the development of pathogenic hyphae in Iregi szürke csíkos and P63LE13 sunflower plants and it was associated with intensive autofluorescence of cells. Both localized and systemic induction of resistance was observed. Importantly, the frequency of mycorrhization of hybrid P63LE13 and PR64H41 was significantly increased upon BTH treatment, so it had a positive effect on the formation of plant-mycorrhiza interactions in sunflower. To our knowledge, this is the first report on the additive effect of BTH on mycorrhization and the positive effect of these inducers against SS in sunflower.