Mutations in Sdh Gene Subunits Confer Different Cross Resistance Patterns to SDHI Fungicides in Alternaria alternata Causing Alternaria Leaf Spot of Almond in California

Alternaria leaf spot caused by Alternaria alternata and A. arborescens is a common disease of almond in California. Succinate dehydrogenase inhibitors (SDHIs) are widely used for its management, however, we observed reduced performance of SDHI fungicides at some field sites. Thus, we evaluated the sensitivity of 520 isolates of the main pathogen A. alternata from major production areas collected between 2006 and 2019 to boscalid and of a subset of 204 isolates to six members of the SDHIs belonging to six sub-groups. Additionally, 97 isolates (14 sensitive and 83 with reduced sensitivity) of the 204 were used to determine the molecular mechanisms of resistance. A wide range of in vitro concentrations to effectively inhibit mycelial growth by 50% (EC50 values) was determined for each fungicide using the spiral gradient dilution method. Some isolates were highly resistant (EC50 values >10 μg/ml) to boscalid (a pyridine-carboxamide), pyraziflumid (a pyrazine-carboxamide), and fluxapyroxad (a pyrazole-4-carboxamide), but not to fluopyram (a pyridinyl-ethyl-benzamide), isofetamid (a phenyl-oxo-ethyl thiophene amide), and pydiflumetofen (a N-methoxy-(phenyl-ethyl)-pyrazole-carboxamide). There was no strong cross resistance among the fungicides tested, including for the two pyrazole-4-carboxamides fluxapyroxad and penthiopyrad (tested for 33 of the 204 isolates). The comparison of EC50 values for fluopyram and isofetamid resulted in the highest coefficient of determination (R2 = 0.582) among ten pairwise comparisons between sub-groups. Sequence analyses of the 97 isolates revealed five mutations in SdhB, SdhC, or SdhD subunits of the Sdh target gene among 73 isolates with reduced sensitivity to at least one SDHI. No mutations were detected in the 14 sensitive isolates and in 10 of the 83 isolates with reduced sensitivity. The most common mutation (59 isolates) was H134R in SdhC. Other mutations included H277Y (8 isolates) and H277L (2 isolates) in SdhB, as well as G79R (2 isolates) and S135R (2 isolates) in SdhC. Mutations H277Y in SdhB and S135R in SdhC were only present in isolates collected in 2012 or earlier. Both conferred mostly high levels of resistance to boscalid and also reduced sensitivity to pyraziflumid, fluxapyroxad, and isofetamid with intermediate EC50 levels. Mutations H277L in SdhB, as well as H134R and G79R in SdhC, that were found in isolates obtained after 2012 had very similar resistance phenotypes with different levels of resistance to boscalid, pyraziflumid, and fluxapyroxad, whereas sensitivity to fluopyram, isofetamid, and pydiflumetofen was mostly less affected. Our data for SDHI fungicides do not support the classical concept of positive cross resistance within a single mode of action. Because some mutations conferred resistance to multiple SDHI sub-groups, however, resistance management needs to consider all SDHIs as a homogenous group that should be mixed or rotated with other modes of action prior to resistance development to either mode of action.

Alternaria leaf spot of broccoli caused by Alternaria alternata in Bangladesh

This study aimed to isolate and characterise the pathogen associated with Alternaria leaf spot on broccoli and to evaluate the inhibitory effects of fungicides against it. We isolated and identified the fungal pathogen as Alternaria sp. using morphological and cultural methods. Based on the aligned sequences of the internal transcribed spacer (ITS) and molecular phylogenetic analysis by the neighbour-joining method, the isolates (Ab1 and Ab2) were confirmed as Alternaria alternata. The conidia of the isolates were dark brown, cylindrical, obclavate to muriform. The conidiophores were olivaceous brown, septate, and branched. The conidial morphology of the isolates ranged from 52.4–92.4 × 10–20 μm with 2–6 transverse and 0–3 longitudinal septa. Both isolates yielded positive results in the pathogenicity test on broccoli leaves by developing brown and circular spots with concentric rings on the leaves surrounded by yellow halos. The culture studies revealed that the maximum growth of the pathogen was obtained at 30 °C and pH 6.0. Tilt 250 WC showed the highest potential in suppressing the mycelial growth of the A. alternata in vitro at a concentration as low as 50 µg/mL. The results from this study contributed to the positive identification of the pathogen and characterised A. alternata as a destructive pathogen of broccoli which may be successfully controlled by the fungicide Tilt.

Response of Intermediate Ripening Tobacco (Nicotiana tabacum) Cultivars to Frog Eye (Cercospora nicotianae) and Alternaria Leaf Spot (Alternaria alternata) in the Medium Growing Areas of Zimbabwe

Background: Alternaria leaf spot (Alternaria alternata) and Frog eye leaf spot (Cercospora nicotianae) are foliar diseases of tobacco (Nicotiana tabacum) which occur mainly in mature tobacco. Hence, the diseases are often associated with maturity of cured tobacco leaf at the tobacco sales floor in Zimbabwe. Where they occur, they can result in significant reductions in yield and quality of cured tobacco leaf. Methods: A study was carried out in Trewaney, Mashonaland West province in Zimbabwe to investigate the response of newly developed ten flue cured tobacco varieties to the two foliar diseases Alternaria leaf spot (Alternaria alternata) and frog eye leaf spot (Cercospora nicotianae). The experiment was laid out as a randomized complete block design (RCBD) with three replicates. Standard agronomic practices in tobacco farming were followed and disease assessments were done 18 weeks after planting. Result: Significant differences (p less than 0.05) for resistance to alternaria leaf spot were observed with the experimental hybrid T60 showing the highest resistance to the disease with a disease score of 1.6 under a 0-6 disease scale that ranges from 0 (no disease) to 6 (severely affected). However, there were no significant (p greater than 0.05) differences among the varieties for frog eye leaf spot resistance, yield (kg/ha) and quality as measured by the grade index, however, K 326 showed the highest resistance to frog eye leaf spot. All the varieties, however, showed resistance to the two diseases. It was concluded that K 326 was the source of the resistance to frog eye leaf spot in all the experimental hybrids evaluated and that T60 had the highest Alternaria leaf spot resistance obtained from its parents which are both classified as resistant to the disease.

Cultural, Morphological Characterization and Aggressiveness of Alternaria alternata Causing Leaf Spot of Soybean

Background: Alternaria leaf spot caused by Alternaria alternata is one of the most important and destructive disease of soybean causing severe yield loss in all soybean growing areas of southern and eastern part of Rajasthan. Successful management of Alternaria leaf spot is mainly dependent on accurate and efficient detection of pathogen, amount of genetic and pathogenic variability present in pathogen population. The main reason for frequent “breakdown” of effective resistance is the variability that exists in the pathogen population, which necessitates a continual replacement of cultivars due to disease susceptibility. Methods: The twelve fungal isolates randomly were collected from six districts of major soybean growing part of Rajasthan i.e., Udaipur, Chittorgarh, Pratapgarh, Kota, Baran and Jhalawar. The culture was purified single spore techniques. These were then further compared among each other for any variations in cultural characters, colour of colonies, Growth rate, conidial morphology and pathogenic variability. Result: Twelve different isolates of A. alternata were obtained in pure culture and characterized for cultural, morphological variation and aggressiveness of this fungus varied in their cultural characters, colour of colonies, growth rate of isolates, conidial morphology and isolates also exhibited variations in incubation period, latent period, number and size of lesions were produced.

Severidade de Mancha-de-alternária em genótipos de girassol no Cerrado do Distrito Federal / Evaluation of Alternaria leaf spot severity on sunflower in the Cerrado region of the Distrito Federal

Este trabalho teve como objetivo comparar a severidade da Mancha-de-alternária de genótipos de girassol em ambientes do Cerrado do Distrito Federal. Três experimentos foram avaliados, dois na Embrapa Cerrados (Planaltina, DF) e um localizado na Embrapa Cerrados, no Centro de Inovação em Genética Vegetal – CIGV, Fazenda Sucupira, (Riacho Fundo II, DF). Foram feitas análises ao longo do ciclo da cultura com um intervalo de 15 dias, sendo a primeira aos 35 dias após emergência. Cada planta foi analisada em três partes: terço inferior, terço médio e terço superior. Ao final de cada experimento, a área abaixo da curva de progresso da doença (AACPD) foi calculada para cada genótipo. De acordo com os resultados observados, o ambiente de sequeiro na Embrapa Cerrados, que deteve a maior média da severidade da doença, entre os ambientes avaliados. Dentre os genótipos avaliados, MG 360 mostrou menor severidade da doença no sequeiro e AGUARA 06 no irrigado da Embrapa Cerrados. O genótipo G 42 no CIGV, se destacou pela menor severidade do fungo. Houve influência significativa do ambiente nos resultados de severidade da doença, que está estreitamente relacionada com os fatores climáticos como temperatura do ar e umidade relativa do ar. A compreensão dessas condições favoráveis ao desenvolvimento do fungo é essencial quando se avalia a resistência em genótipos de girassol.

Molecular characterization of leaf spot caused by Alternaria alternata on buttonwood (Conocarpus erectus L.) and determination of pathogenicity by a novel disease rating scale

The buttonwood (Conocarpus erectus L.) is a mangrove shrub belonging to Combretaceae family. It mostly grows on the shorelines of tropical and subtropical regions in the world. It was introduced to Lasbela University of Water, Agriculture & Marine Sciences (LUWMS), Uthal, Baluchistan as an ornamental plant as it grows well under harsh, temperate and saline conditions. During a routine survey, typical leaf spot symptoms were observed on the leaves of buttonwood plants. A disease severity scale for alternaria leaf spot of buttonwood was developed for the first time through this study. Disease severity according to the scale was 38.97%. The microscopic characterizations was accomplished for the identification of Alternaria alternata and Koch’s postulates were employed to determine the pathogenicity. For molecular identification, 650 bp internal transcribed spacer (ITS) regions (ITS1, 5.8s and ITS2) were amplified from three representative isolates (LUAWMS1, LUAWMS2 and LUAWMS3) through polymerase chain reaction (PCR). The nucleotide sequences from ITS regions of the isolates were submitted to NCBI with GenBank accession numbers MW585375, MW585376 and MW585377, respectively. The phylogenetic tree of 22 A. alternata isolates was computed and representative isolates exhibited 99.98% genetic similarity with mangroves ecosystem isolates. This study reports the incidence of alternaria leaf spot of buttonwood at LUWMS for the first time. It is suspected that the disease may spread further. Therefore, effective management strategies should be opted to halt the further spread of the disease.

INVESTIGATION OF CORRELATION BETWEEN TRAITS AND PATH ANALYSIS OF SUNFLOWER (HELIANTHUS ANNUUS L.) GENOTYPES

This study was carried out to determine the correlation between agronomic traits and seed yield of sunflower hybrids and also to determine the direct and indirect effects of analysed traits on seed yield. Correlation studies revealed that days to 50 % flowering, plant height, head diameter, volume weight, seed yield per plant and oil content were important selection indices for both oil and seed yield improvement. Alternaria leaf spot severity had negative and significant correlation on seed yield per plant, oil content and oil yield per plant. This indicated the importance of resistance breeding for Alternaria leaf spot disease. The path analysis revealed that traits namely; days to 50 % flowering, head diameter, oil content, plant height and Alternaria leaf spot severity were important selection indices. Though the correlation analysis showed that 100-seed weight and volume weight were important, the path analysis indicated that these traits were less important