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.     

The Influence of Plant Extracts on Phytotoxin Production and Growth Rate of Alternaria helianthi

When liquid cultures of Alternaria helianthi were supplemented with aqueous extracts of leaf tissue of its host plant (sunflower), pronounced effects on both growth and production of the toxin deoxyradicinin were apparent. Very low levels of leaf extract stimulated toxin production but did not significantly affect growth, while higher levels markedly stimulated mycelial growth and at the same time toxin production was greatly suppressed. Leaf extracts of non-host plant species were also stimulatory to phytotoxin biosynthesis.     

Deoxyradicinin and 3-epideoxyradicinol production by the sunflower pathogen Alternaria helianthi

The phytotoxic compound deoxyradicinin, a metabolite of Alternaria helianthi, was isolated from naturally infected leaves of sunflower (Helianthus annuus). The production of this toxin in liquid culture together with 3-epideoxyradicinol is discussed. Production of deoxyradicinin by liquid cultures was observed to decrease after successive sub-culture of the fungus on solid medium. All 10 accessions of Helianthus tested were sensitive to deoxyradicinin although some quantitative differences between lines were noted. Evidence is presented for the metabolism of deoxyradicinin by host leaf tissue.     

A greenhouse assay to screen sunflower for resistance to Alternaria helianthi

A greenhouse assay to screen sunflower for resistance to Alternaria helianthi is described. A comparison of conditions led to the following standard conditions being recommended. The first or second pair of leaves of seedling plants at the V8 growth stage are inoculated using inoculum grown on sunflower leaf extract agar for 5–10 days at an inoculum density of 1–2 spores cm² of leaf tissue. A 48 h dew period should be applied to plants covered by a plastic tent. A dew period temperature of 26/26°C night/day and a post-dew period temperature relative to that experienced under local growing conditions should be applied. Lesions are measured 7 days after inoculation, and mean lesion size per plant is calculated. Mean lesion size of lines being tested is expressed as a proportion of the mean lesion size of a susceptible standard included in each screening experiment.     

Specific PCR-based detection of Alternaria helianthi: the cause of blight and leaf spot in sunflower

Alternaria helianthi is an important seed-borne pathogenic fungus responsible for blight disease in sunflower. The current detection methods, which are based on culture and morphological identification, are time-consuming, laborious and are not always reliable. A PCR-based diagnostic method was developed with species-specific primers designed based on the sequence data of a region consisting of the 5.8S RNA gene and internal transcribed spacers—ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of A. helianthi. The specificity of the primer pairs AhN1F and AhN1R designed was verified by PCR analysis of DNA from 18 Alternaria helianthi strains isolated from India, 14 non-target Alternaria spp. and 11 fungal isolates of other genera. A single amplification product of 357-bp was detected from DNA of A. helianthi isolates. No cross-reaction was observed with any of the other isolates tested. The detection limit of the PCR method was of 10?pg from template DNA. The primers could also detect the pathogen in infected sunflower seed. This species-specific PCR method provides a quick, simple, powerful and reliable alternative to conventional methods in the detection and identification of A. helianthi. This is the first report of an A. helianthi-specific primer set.     

Melanin Biosynthesis Inhibitors from Tarragon Artemisia dracunculus

The EtOH extract of tarragon Artemisia dracunculus, a perennial herb in the family Asteraceae, was found to potently inhibit α-melanocyte-stimulating hormone (α-MSH) induced melanin production in B16 mouse melanoma cells. Bioassay-guided fractionation led to the isolation of two alkamide compounds, isobutyl (1) and piperidiyl (2) amides of undeca-2E,4E-dien-8,10-dynoic acid. The respective EC₅₀ values for melanin biosynthesis inhibition were 1.8 and 2.3 μg/mL for 1 and 2.     

Growth promotion activity and biological control for the management of leaf blight incited by Alternaria helianthi

The Pseudomonas fluorescens (Pf1), Bacillus subtilis (Bs1) are the major potential biocontrol agents against foliar pathogens. MPf1 and MBs1 were found to be the most effective in inhibiting the mycelial growth of Alternaria helianthi. These biocontrol agents have the maximum capacity in controlling the spore germination, and data showing the growth-promoting effect of biocontrol agents and inhibition of seed-borne fungi are available. Seed-borne infections of A. helianthi are controlled by seed treatment with P. fluorescens, which showed least seed infection. The root length and shoot length has also been increased.     

暹罗炭疽菌同源异型盒转录因子CsHtf1的生物学功能

【背景】暹罗炭疽菌(Colletotrichum siamense)是橡胶炭疽病害的主要致病菌,严重制约着天然橡胶产量。在植物致病真菌中广泛存在同源异型盒转录因子,其参与调控真菌无性生殖、侵染和代谢等诸多方面。【目的】明确在暹罗炭疽菌中鉴定的一个同源异型盒转录因子CsHtf1的生物学功能。【方法】利用同源重组的方法获得Cshtf1基因的敲除突变株,并对其营养生长、孢子产生和致病性等表型进行分析。【结果】Cshtf1基因编码600个氨基酸且含有1个HOX结构域;与野生型相比,Cshtf1敲除突变株营养生长和致病性无显著差异,而突变株分生孢子产量显著降低且黑色素产量增加。【结论】CsHtf1参与调控暹罗炭疽菌的分生孢子及黑色素产生。     

Bacterial compounds,as biocontrol agent against early blight (Alternaria solani) and tobacco cut worm (Spodoptera litura Fab.) of tomato (Lycopersicon esculentum Mill.)

Abstract

Tomato, the important vegetable crop faces yield loses by foliar disease, Early Blight caused by Alternaria solani and infestation by tobacco cut worm, Spodoptera litura. Synthetic pesticides used for disease and pest control resulted in environmental pollution as well as development of resistance. By way of a need to develop a new biopesticide, bacteria were tested for their anti-fungal and insecticidal activity. Volatile compounds and peptides from the bacteria, Bacillus subtilis, Staphylococcus aureus and Pseudomonas aeruginosa were able to inhibit the hyphal growth and melanin production of A. solani. S. aureus showed the highest inhibitory effect as well as reduced the disease severity. B. subtilis exhibited the highest mortality rate of 87% and 83% against I and II instar of S. litura, respectively. The bacteria offered effective biocontrol at 35?°C. Bioactive substances from the bacteria can be used as potential biocontrol agents against the pest and disease of tomato.     

Deoxyradicinin,a novel phytotoxin from Alternaria helianthi

A novel major metabolite which possesses phytotoxic and antifungal properties has been isolated from culture filtrates of Alternaria helianthi and its structure elucidated as deoxyradicinin.     

Phenols displaying tyrosinase inhibition from Humulus lupulus

Tyrosinase is the rate-limiting enzyme for the production of melanin and other pigments via the oxidation of l-tyrosine. The methanol extract from Humulus lupulus showed potent inhibition against mushroom tyrosinase. The bioactivity-guided fractionation of this methanol extract resulted in the isolation of seven flavonoids (1–7), identified as xanthohumol (1), 4′-O-methylxanthohumol (2), xanthohumol C (3), flavokawain C (4), xanthoumol B (5), 6-prenylnaringenin (6) and isoxanthohumol (7). All isolated flavonoids (1–7) effectively inhibited the monophenolase (IC₅₀s?=?15.4–58.4?µM) and diphenolase (IC₅₀s?=?27.1–117.4?µM) activities of tyrosinase. Kinetic studies using Lineweaver–Burk and Dixon-plots revealed that chalcones (1–5) were competitive inhibitors, whereas flavanones (6 and 7) exhibited both mixed and non-competitive inhibitory characteristics. In conclusion, this study is the first to demonstrate that the phenolic phytochemicals of H. lupulus display potent inhibitory activities against tyrosinase.     

Ethanolic extract of mango (Mangifera indica L.) malformed inflorescence as effective antifungal agent

Mango (Mangifera indica L.) suffers from floral and vegetative malformation and crop production is seriously affected. The anti-fungal activity of ethanolic extract of malformed mango inflorescence was observed at different concentrations (1000, 2000, 3000, 4000 and 5000 μl/ml) against 10 fungi, viz., Ustilago cynodontis, Cercospora cajani, Sphaerotheca sp., Cercospora sp., Alternaria solani, Bipolaris sp., Helminthosporium sp., Curvularia sp., Fusarium udum and Alternaria cajani. Spore germination of most of the fungi was inhibited at 5000 μg/ml. Some of them were also susceptible at 3000 or 4000 μg/ml concentration. Analysis of phenolic acids by high performance liquid chromatography (HPLC) showed 18 peaks in the extract, but only four could be identified, viz., capachin, gallic, benzoic and cinnamic acids. Because of the high efficacy of ethanolic extract of malformed mango inflorescence, its use under field conditions to control some plant diseases has been suggested.     

3-Epideoxyradicinol and the biosynthesis of deoxyradicinin

A novel phytotoxic compound, in addition to the known phytotoxin deoxyradicinin, has been isolated from Alternaria helianthi and its structure elucidated as 3-epideoxyradicinol. Its epimer is also briefly described. The polyacetate biosynthetic origin of deoxyradicinin has been demonstrated.     

Inhibitory Effects of Bakuchiol,Bavachin, and Isobavachalcone Isolated from Piper longum on Melanin Production in B16 Mouse Melanoma Cells

An EtOH extract of fruits of Piper longum was found to exhibit a potent inhibitory effect against α-melanocyte-stimulating hormone (α-MSH)-induced melanin production in B16 mouse melanoma cells. Bioassay-directed fractionation led to the isolation of prenylated phenolic compounds bakuchiol, bavachin, and isobavachalcone. These compounds and the crude extract of the fruits of P. longum may have suppressive effects against pigmentation by melanin in the skin.     

Analysis of Cultural and Genetic Diversity in Alternaria helianthi and Determination of Pathogenic Variability Using Wild Helianthus Species

The study aims at assessment of morphological, molecular and pathogenic variability of Alternaria helianthi, incitant of leaf blight of sunflower. Morphological characteristics determined for 26 isolates of A. helianthi from India revealed variations in shape of culture, pigmentation, conidial measurements, number of septa and colony growth. The conidia of isolate Ah‐7 were long, while conidia of isolate Ah‐15 were short. Based on cultural characters, isolates were classified into four groups. Genetic variability of the isolates was assessed by random amplified polymorphic DNA analysis. Good polymorphism was observed and cluster analysis indicated presence of six genetically distinct groups among the isolates. The isolates Ah‐1, Ah‐7 and Ah‐14 were genetically distinct. Resistant sources are not available in cultivated sunflower, while wild Helianthus species possess resistance to multiple stresses. We evaluated reaction of wild Helianthus species to isolates of A. helianthi. Among wild Helianthus species, H. tuberosus followed by H. occidentalis showed moderately to highly resistant reaction to all the isolates and recorded less disease incidence. The species H. argophyllus followed by H. laevigatus showed more disease incidence. The cultivated sunflower recorded susceptible reaction to most of the isolates and recorded high disease incidence. The isolates differed significantly for pathogenic reaction and were grouped into three pathogenicity groups; low, medium and high. Six isolates induced <20% disease incidence and were included in the low pathogenicity group. Majority of isolates were in the medium pathogenicity group. Six isolates i.e. Ah‐9, Ah‐10, Ah‐18, Ah‐20, Ah‐24 and Ah‐26 induced more than 50% disease incidence and were considered high pathogenicity group. Our results demonstrate the existence of considerable variation in resistance of Helianthus species to A. helianthi and also in morphological and genetic characters of A. helianthi isolates prevalent in India.     

The Nature of Melanin Pigments of Several Micro- and Macromycetes

New inhibitors of melanin formation by micromycetes Aspergillus carbonarius, Alternaria alternata, and Paecilomyces variotii and basidiomycetes Inonotus obliquus and Phellinus robustus were found. Precursors of melanin pigments were isolated and identified. p-Hydroxybenzoic acid was identified among the products of alkaline degradation of melanin formed by micromycetes, whereas in the case of macromycetes this was protocatechuic acid. Therefore, melanins of the former were found to belong to the dihydronaphthalene group, whereas those of the latter belong to catechols.     

梨果黑斑病菌互隔交链孢(Alternaria alternata) hnr基因的克隆及其对侵染结构分化的调控

【背景】羟基萘还原酶(hydroxynaphthalene reductase,HNR)是1,8-间苯二酚(1,8-dihydroxynaphthalene,DHN)黑色素合成途径中起关键作用的酶,研究表明HNR不仅参与真菌黑色素合成,而且对其生长发育及致病性也具有一定的调控作用,但HNR对真菌病原物侵染结构分化的调控研究鲜见报道。【目的】在对梨果黑斑病菌互隔交链孢(Alternaria alternata) HNR的基因进行克隆与生物信息学分析的基础上,通过药理学方法初步探讨HNR对A.alternata生长及侵染结构分化的调控作用,为进一步揭示HNR在A.alternata侵染结构分化形成过程中的分子机制提供理论依据。【方法】对梨果黑斑病菌A.alternata的2个hnr基因进行了克隆;通过gene structure display server、open reading frame (ORF) Finder及conserved domain search等数据库及相关软件,对hnr基因及蛋白进行生物信息学分析,并利用HNR特异性抑制剂三环唑处理分析其对A.alternata生长发育、黑色素合成和侵染结构形成的影响,同时采用实时荧光定量PCR (RT-qPCR)技术分析了hnr基因在A.alternata不同侵染结构分化时期的表达特性。【结果】从梨果黑斑病菌A.alternata克隆得到2个羟基萘还原酶基因hnr的编码区全长,分别命名为Aa4hnr和Aa3hnr,其中Aa4hnr基因全长为1 266 bp,编码了268个氨基酸,无内含子,有9个ORF;Aa3hnr基因全长为1 356 bp,编码了267个氨基酸,含有2个大小分别为51 bp和49 bp的内含子,有17个ORF;进化分析表明,Aa4hnr和Aa3hnr与Ophiobolus disseminans和Alternaria arborescens分别具有较高的一致性,同时Aa4hnr和Aa3hnr编码的蛋白均含有NAD (P)结合域,属于短链脱氢酶/还原酶(short-chain dehydrogenase/reductase,SDR)家族。药理学结果表明,三环唑处理显著降低了A.alternata DHN黑色素的生物合成,抑制了疏水性诱导的A.alternata侵染结构的形成;进一步分析Aa4hnr和Aa3hnr在疏水表面诱导的A.alternata孢子萌发阶段(2 h)、附着胞形成阶段(6 h)、侵染菌丝形成阶段(8 h)的基因表达量,Aa4hnr的基因表达量在A.alternata侵染结构分化的各个时期均发生下调,Aa3hnr在附着胞形成阶段(6 h)表达量下调,然而在侵染菌丝形成阶段(8 h)显著上调表达。【结论】Aa4hnr和Aa3hnr对梨果黑斑病菌侵染具有一定的调控作用。     

Phytotoxins from Alternaria helianthi: radicinin,and the structures of deoxyradicinol and radianthin

A novel compound, radianthin, with phytotoxic activity was isolated from liquid cultures of Alternaria helianthi and identified as a pyrone related to radicinin. A second metabolite was identified as radicinin itself while deoxyradicinol is described for the first time as a natural product.