Antifungal activity of polyacetylenes isolated from Cirsium japonicum roots against various phytopathogenic fungi

Antifungal substances from a methanol extract of Cirsium japonicum roots were purified and characterized, and their antifungal activities against various plant pathogens were evaluated. Three polyacetylene substances were isolated from roots of C. japonicum using repeated column chromatography; these were identified as ciryneol A, ciryneol C and 1-heptadecene-11,13-diyne-8,9,10-triol by mass and nuclear magnetic resonance spectral analyses. In vitro antifungal activity of the three substances varied according to compound and target species. Magnaporthe oryzae, Colletotrichum coccodes, Colletotrichum acutatum, Pythium ultimum and Botrytis cinerea were relatively sensitive to the three polyacetylenes, with IC₅₀ values below 50 μg mL⁻¹. In vivo, they all showed similar and broad antifungal spectra against the seven plant diseases tested. At 500 μg mL⁻¹, all three compounds effectively suppressed the development of rice blast, rice sheath blight, tomato late blight, wheat leaf rust and red pepper anthracnose, with control values over 90%. They were highly active especially against wheat leaf rust; they controlled the development of this disease more than 88% even at a concentration of 125 μg mL⁻¹. In addition, ciryneol C effectively suppressed barley powdery mildew. This is the first report on the antifungal activities of the three polyacetylenes from roots of C. japonicum against plant pathogenic fungi. Polyacetylenes from roots of C. japonicum may contribute to the development of environmentally safer alternatives to protect crops from various phytopathogenic fungi.     

Multiple effects of two drivers of agricultural change,labour shortage and water scarcity,on rice pest profiles in tropical Asia

The effects of shortage of agricultural labour, represented by direct-seeding technologies replacing transplanting, and of water scarcity, represented by limited water supply, on rice pests (pathogens, weeds, insects) were studied. The analysis was based on a field characterisation data set covering six sites in tropical Asia and several cropping seasons. Two-way ANOVAs and MANOVA were, respectively, used to test the effects of the two factors on individual rice pest injuries, and on the profile of injuries that an individual rice crop experiences over a cropping season. Fourteen injuries were considered, which individually were significantly (P < 0.05) influenced by crop establishment (nine injuries), by water management (11 injuries), or their interaction (nine injuries). Multiple analysis of variance indicated that rice injury profiles, regionally, are altered by both factors. The results suggested that injuries caused by some pests would be enhanced (e.g., brown spot, planthoppers) or reduced (e.g., stem rot, sheath blight, rice whorl maggot) by crop establishment shifting from transplanting to direct-seeding. Water shortages, proxied in our data by poor water management, would favour some injuries (e.g., sheath rot, brown spot, neck blast, whiteheads) or suppress others (e.g., stem rot, sheath blight). Very strong increases in weed infestation were observed with both direct-seeding and poor water management. The results are discussed with respect to pest management in rice.     

Anti-Rhizoctonia solani activity by Desmos chinensis extracts and its mechanism of action

The objectives of this study were to investigate the mechanisms of action and to evaluate the potential application of Desmos chinensis extracts for controlling rice sheath blight. The dichloromethane extract from D. chinensis demonstrated high antifungal activity against Rhizoctonia solani. The extract was shown to have anti-R. solani activity with minimum inhibitory concentration (MIC) and minimum fungicidal concentration values ranging from 31.2 to 62.5 μg mL⁻¹ and from 62.5 to 500 μg mL⁻¹, respectively. Bioautography on thin-layer chromatography plates demonstrated antifungal activity of the extract with an R_f value of 0.33. A total of 7 compounds, 2 benzoate esters (benzyl benzoate and benzyl 2-hydroxybenzoate), 2 sesquiterpenoids (α-eudesmol and β-eudesmol), 1 aromatic alcohol (benzyl alcohol), 1 aromatic ketone (acetophenone) and 1 diterpenoid (phytol), were detected and identified using gas chromatography–mass spectrometry analysis. Electron micrographs confirmed the effects of the extract on morphological and ultrastructural alterations in the treated fungal cells. The micrographs of the mycelia treated with the extract at 4MIC illustrated aberrant morphology such as shrinkage, partial distortion and globular structures of different sizes along the surface of the mycelia. Damaging membranous structures including disruption of the cell membrane, partial loss of nuclear membranes, and depletion of hyphal cytoplasm and membranous organelles were observed. Foliar application of D. chinensis extract at a concentration of 2 mg mL⁻¹ on rice markedly decreased sheath blight severity. It was concluded that the application of D. chinensis extract can be used as a botanical fungicide to control rice sheath blight.     

辽河三角洲水稻纹枯病菌丝融合群测定

辽河三角洲是辽宁省水稻主产区,水稻纹枯病近年来持续严重发生,本研究对辽河三角洲稻区的不同水稻品种上分离获得的纹枯病菌菌株进行融合测试,以便探明水稻品种来源各异的病菌菌群类别及其分布的关系,研究探讨辽河三角洲水稻纹枯病菌菌丝融合群构成,为生产上病害流行、预测以及针对性进行水稻品种选育和推广应用,提供理论基础。     

Comprehensive Characteristics of MicroRNA Expression Profile Conferring to Rhizoctonia solani in Rice

MicroRNAs(miRNAs) are about 22 nucleotides regulatory non-coding RNAs that play versatile roles in reprogramming plant responses to biotic and abiotic stresses. However, it remains unknown whether miRNAs confer the resistance to necrotrophic fungus Rhizoctonia solani in rice. To investigate whether miRNAs regulate the resistance to R. solani, we constructed 12 small RNA libraries from susceptible and resistant rice cultivars treated with water/pathogen at 5 h post inoculation(hpi), 10 hpi and 20 hpi, respectively. By taking the advantage of next-generation sequencing, we totally collected 400–450 known mi RNAs and 450–620 novel miRNAs from the libraries. Expression analysis of mi RNAs demonstrated different patterns for known and novel miRNAs upon R. solani challenge. Thirty-four mi RNA families were identified to be expressed specifically in rice, and most of them were involved in plant disease resistance. A particular Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis result revealed that a great majority of target genes of regulated miRNAs belonged to the pathway of plant-pathogen interaction. Moreover, miR444 b.2, miR531 a, mir1861 i, novel_miR1956 and novel_miR135 conferred response to R. solani infection confirmed by Northern blot. Our global understanding of miRNA profiling revealed that the regulation of mi RNAs may be implicated in the control of rice immunity to R. solani. Analysis of the expression of miRNAs will offer the community with a direction to generate appropriate strategies for controlling rice sheath blight disease.     

Occurrence of rice blast (Magnaporthe oryzae) and identification of potential resistance sources in Uganda

Rice blast caused by Magnaporthe oryzae continues to be the most destructive disease of rice worldwide, and is a number one disease of rice in Uganda. We present the footprints of M. oryzae importance, distribution, incidence and severity in the rice growing agro-ecologies of Uganda for 2009/2010, and the potential mitigation measures. Our data show that rice blast affects more than 50% of the cultivated rice area on average, and ranks as the most important disease encountered in the field. Between and within agroecologies, both incidence and severity varied (P < 0.05) reflecting the contribution of different cropping practices on rice blast epiphytotics. The highest blast incidence and severities were recorded in Bugiri, Butaleja, Mbale and Lira farmlands, which are the ancestral rice cultivation areas in Uganda. These areas showed mean yield reductions of >30% relative to other locations, suggesting that rice production history played a significant role in rice blast outbreaks. Broadcasting and drill seeding yielded 42.4% less than transplanted rice. Growing two rice crops per year is one of the methods suggested to increase rice production in Uganda, but there was a higher disease incidence in the late season than in the early season, indicating the need for improved resistant varieties. Four blast resistance genes (Pi9, Piz-t, Pi19 and Piz-5) and the cultivar Tetep had the lowest (≤4) blast severity scores in all the test locations. It could be suggested that these genes are potential resistance sources for developing varieties, which would be more relevant for the double cropping systems.     

水稻花器介导法转目标基因的研究

研究和分析了受体品种、质粒浓度对水稻花器介导法转化的影响。供试的20个水稻品种（系）中有9个适用于质粒转化,不同受体品种要求的质粒适宜浓度不同。经潮霉素（hygromycin）筛选、PCR分析、抗稻纹枯病和稻瘟病鉴定、GUS活性检测转基因水稻后代,验证了水稻花器介导法转目标基因的有效性。获得了含细菌几丁质酶基因的优丰162转基因T4 代,它表现出对叶瘟病和纹枯病抗性有明显提高。     

Resistance responses of rice to rice blast fungus after seed treatment with the endophytic Achromobacter xylosoxidans AUM54 strains

In the present investigation, we studied resistance imparted by seed treatment with an endophytic strain of Achromobacter xylosoxidans, AUM54, against rice blast caused by Magnaporthe oryzae. In vitro studies showed that A. xylosoxidans AUM54 was able to inhibit mycelial growth of M. oryzae by 11% and was able to increase rice germination and seedling vigor index of rice by 31 and 114%, respectively. AUM54 also showed better survivability in the spermosphere and spermoplane and was able to move systemically through the roots and stem. Among the evaluated carriers, liquid formulation amended with 2% glycerol sustained the maximum bacterial population (7.4 log cfu ml⁻¹) after six-months-storage at room temperature. Plants treated with A. xylosoxidans AUM54 followed by inoculation with M. oryzae showed a significant increase in the activities of defense related enzymes such as polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia-lyase (PAL) and chitinase. A. xylosoxidans AUM54 treatment was able to reduce blast disease incidence by 39% in treated rice plants. Additionally, inoculation with A. xylosoxidans AUM54 significantly enhanced the growth (3–13% plant height), and yield (11–31%) of inoculated rice plants under no-disease and disease conditions in the greenhouse experiments.     

Foliar Application of Benzothiadiazole and Salicylic Acid to Combat Sheath Blight Disease of Rice

A field study was undertaken to ascertain the effects of elicitors viz. benzothiadiazole (BTH) and salicylic acid (SA) on defense related enzymes viz. peroxidase, phenylalanine ammonia lyase, superoxide dismutase, chitinase and β-1,3-glucanase, and phenols in rice (Pusa Basmati I) plants. First foliar spray of BTH (50 mg/kg) and SA (50 mg/kg) was done at the maximum tillering stage and inoculation with Rhizoctonia solani was carried 24 h after elicitor treatment. Elicitors were further sprayed at every growth stage. Time course analysis showed peak accumulation of defense related enzymes and phenols in the rice leaves treated with BTH and SA, and accumulation was the highest at the flowering stage. Higher enzymatic activity was observed in elicitor treated plants inoculated with R. solani. Compared to the untreated control plants, application of elicitors before R. solani inoculation significantly elicited the defense related enzymes and phenols. Moreover, application of elicitors had a positive effect on yield and disease reduction. It is suggested that pretreatment of rice leaves with BTH and SA could be used to enhance the level of protection against sheath blight and to improve rice yield in the fields.     

Marker-Assisted Breeding of Thermo-Sensitive Genic Male Sterile Line 1892S for Disease Resistance and Submergence Tolerance

Rice line 1892S is an elite thermo-sensitive genic male sterile(TGMS)line for two-line hybrid rice production.However,1892S is susceptible to rice blast,bacterial blight and submergence.Here we reported the introduction of blast resistance(R)gene Pi9,bacterial blight R gene Xa21 and submergence tolerance gene Sub1A into 1892S genetic background through backcrossing and marker-assisted selection.The improved TGMS line 31892S and its hybrids conferred disease resistance to rice blast and bacterial blight,and showed submergence tolerance for over 14 d without significant loss of viability.The sterility-fertility conversion of 31892S was similar to that of 1892S.31892S and its derived hybrid rice had similar agronomic traits and grain quality with 1892S and the control hybrid rice,respectively.The newly developed 31892S provided an improved TGMS line for two-line hybrid rice production with disease resistance to rice blast and bacterial blight,and submergence tolerance with no yield penalty or change in grain quality.     

Detection and characterization of bismerthiazol-resistance of Xanthomonas oryzae pv. oryzae

The resistance to bismerthiazol in the Xanthomonas oryzae pv. oryzae population in China has not been clearly determined. Three bismerthiazol-resistant mutants (1-1-1, 2-1-1, 4-1-1) of X. oryzae pv. oryzae were induced on rice plants and the characteristics were compared with their parental strain ZJ173. After 10 subcultures on fresh bismerthiazol-free nutrient agar plates and after being inoculation onto and re-isolation from untreated rice plants two times, the mutants remained pathogenicity on rice plants treated with 300 μg/ml bismerthiazol, indicating that the in vivo-induced bismerthiazol resistance was stable in vivo. The in vitro characteristics of the bismerthiazol-resistant mutants and a wild-type bismerthiazol-sensitive strain (ZJ173) were also compared. The three mutants grew faster than ZJ173 during the logarithmic growth phase but at the same rate during the decline phase. Bismerthiazol had protective and curative activity against strain ZJ173 but not against the three mutants. To evaluate the current degree of bismerthiazol resistance in field populations of X. oryzae pv. oryzae in China, 781 isolates of X. oryzae pv. oryzae were obtained from 1419 leaf samples collected from Yunnan, Sichuan, Jiangsu, Anhui, Hubei, Guangdong, Hainan, and Hunan Provinces in China from 2007 to 2009. The in vivo sensitivity of 505 of the 781 isolates to bismerthiazol was determined by applying them to rice plants treated with 300 μg/ml bismerthiazol and evaluating blast severity. The tested strains were considered resistant, if they showed <70% inhibition relative to strain ZJ173. Of the 505 isolates, 62 were resistant to bismerthiazol. The resistance frequencies among field population ranged from 0 to 21.1%, differed among the provinces, and was 11.2, 20.0 and 10.4% in 2007, 2008 and 2009, respectively. Resistance determined in vivo was not related to resistance determined in vitro. The in vivo-induced bismerthiazol resistance identified in this study will serve as a base line for further studying bismerthiazol resistance in fields, and the current degree of bismerthiazol resistance in field populations of X. oryzae pv. oryzae in China determined would be useful for the rice growers for control of rice bacterial blight.     

稻秆还田对水稻主要病害发生的影响

通过水稻秸秆还田小区试验,初步探讨了多年秸秆还田对15个水稻品种病害的影响。试验结果表明：秸秆还田的老稻田15个水稻品种的稻瘟病、鞘腐病、纹枯病的病情指数都高于对照稻田的病情指数。黑龙江省农垦总局建三江分局主栽品种空育131,叶瘟病情指数增加了2．3％,穗颈瘟增加了2．6％,鞘腐病增加了2．2％,纹枯病增加了0．4％。     

Suppression of rice sheath blight disease using a heat stable culture filtrate from Streptomyces philanthi RM-1-138

Streptomyces philanthi RM-1-138, isolated from the rhizosphere soil of chili pepper in southern Thailand, was investigated for its antagonistic activity against phytopathogenic fungi. In dual culture on glucose yeast-malt extract (GYM) agar plates, this strain suppressed the mycelial growth of all seven plant pathogenic fungi tested (Rhizoctonia solani PTRRC-9, Pyricularia grisea PTRRC-18, Colletotrichum gloeosporioides NBCRSR-3, Colletotrichum capcisi NBCRSR-15, Ganoderma boninense NBCRSR-26, Fusarium fujikuroi PTRRC-16 and Bipolaris oryzae PTRRC-36) with an 82.2–89.2% inhibition and that was most pronounced on R. solani PTRRC-9. Heat treatment of the culture filtrate from growing R. solani PTRRC-9 at 40 °C, 60 °C, 80 °C, and 100 °C for 30 min and 121 °C for 15 min had no negative effect on the inhibitory activity against R. solani PTRRC-9 tested on both solid and liquid culture. The effective dose (>80% inhibition) of culture filtrate in liquid culture was at 5.0% (v/v) while it was at 10% (v/v) on the solid medium. This effectiveness was similar to those of the four chemical fungicides tested. The effect of S. philanthi RM-1-138 against R. solani PTRRC-9 was investigated using SEM and TEM. The compounds produced by S. philanthi RM-1-138 induced alterations to the cell-wall structure of R. solani PTRRC-9, that resulted in the loss of cytoplasm materials by partial lysis. The greenhouse experiment revealed that using either the culture filtrate or the autoclaved culture filtrate from S. philanthi RM-1-138 effectively suppressed rice sheath blight disease by up to 65.6 and 60.8%, respectively.     

Fusarium solani Upregulated Sesquiterpene Synthase Expression,Sesquiterpene Production and Allelopathic Activity in Piper betle L.

Activators of sesquiterpene synthase (STS) gene expression and sesquiterpene production in Piper betle L. were examined using quantitative real time PCR and gas chromatography mass spectrometry methods, and the allelopathic activity of untreated and Fusarium solani-treated betel extracts was tested on seed germination and on the shoot and root growth of Thai rice variety PSL2 (Oryza sativa cv. Phitsanulok 2) and three dominant paddy weeds (Eclipta prostrata, Echinochloa crus-galli and Chloris barbata). The results demonstrated that F. solani dramatically upregulated STS expression and productions of β-cubebene, β-caryophyllene and germacrene D sesquiterpene when compared with the untreated control, and that betel extracts had a greater inhibitory effect on weeds than on rice. The effects were more clearly detected on seed germination and root growth than on shoot growth, and they were found to be dose-dependent. It is also noted that F. solani-treated extract had stronger effects than the untreated extract. The species most sensitive to the allelopathic effects was C. barbata, germination of which was completely inhibited even at a dose of 0.1 mg/mL untreated extract. With regards to rice, although betel extract at 1.0 mg/mL showed no inhibition on germination, it affected the elongation of rice roots, in addition to those of the tested weeds. The obtained data suggested that F. solani has potential as an activator of sesquiterpene allelochemical production via STS expression, the latter leading to the treated betel extract having a stronger phytotoxic effect. These results were beneficial in the promotion of natural herbicide production using biotechnology.     

Metabolite profiling of sheath blight disease resistance in rice: in the case of positive ion mode analysis by CE/TOF-MS

Rice sheath blight is an important disease caused by Rhizoctonia solani. The resistant and susceptible rice lines (32R and 29S, respectively) showed different responses to R. solani infection in metabolite levels. The aim of this study was to characterize the metabolite levels in rice lines during R. solani infection using capillary electrophoresis equipped with time of flight mass spectrophotometry (CE/TOF-MS) in positive ion mode. Hundred metabolites were identified and classified into six clusters by hierarchical cluster using Mass Profiler Professional software. Changes in metabolite level at inoculated 32R and 29S were mapped on branches of tricarboxylic acid and glycolysis pathway. Volcano plot successfully filtered the metabolites based on fold change and p-value. The volcano plot result showed that 10 metabolites were up and down regulated in inoculated 32R relative to 29S. One metabolite, chlorogenic acid, showed a positive response in 32R. Meanwhile, pipecolic acid showed as the highest magnitude of fold change and p-value significance level in 29S. In addition, eight amino acids; glutamate, γ-aminobutyric acid, glycine, histidine, phenylalanine, serine, tryptophan, and tyrosine showed increase in 29S after R. solani inoculation.     

新型生防农药0.4%低聚糖素防治水稻病害试验报告

通过对比试验,了解新型生防农药0.4%低聚糖素防治水稻病害情况。结果表明:0.4%低聚糖杀菌剂能较好的防治水稻稻瘟病、叶鞘腐败病和水稻纹枯病等细胞壁为甲壳质的真菌性病害。使用量以150～200ml/667m2为宜。     

Biocontrol of rice blast by the phenaminomethylacetic acid producer of Bacillus methylotrophicus strain BC79

Strain BC79, isolated from primeval forest soil in Qinling, Mountains, China, was identified as Bacillus methylotrophicus based on morphological, biochemical, physiological and chemotaxonomic analyses as well as phylogenetic 16S rDNA sequencing data. This strain was able to suppress mycelial growth and conidial germination of numerous plant pathogenic fungi in dual cultures on solid media. For exploring potential biocontrol activity, we assessed fermentation conditions for studying B. meth1ylotrophicus BC79. The active substance of BC79, phenaminomethylacetic acid, was extracted by TLC and HPLC, and identified as the strongest inhibitory substance described in B. methylotrophicus. Experiments in a greenhouse showed that application of BC79 culture filtrates 24 h before inoculation of Magnaporthe oryzae, the causal agent of rice blast, had 89.87% biocontrol efficiency. B. methylotrophicus BC79 colonized rice plant tissues and at 10 days after filtrate application, its population in leaves (1.65 × 10⁸ CFU/g) was much larger than in stems (6.78 × 10⁷ CFU/g) or roots (3.56 × 10⁷ CFU/g). Field trials indicated that BC79 culture filtrate (4000 g/667 m²) showed the highest efficiency for M. oryzae, with 84.8% biocontrol effect, followed by of 15% phenaminomethylacetic acid extract (75.5%) and 20% tricyclazole (76.1%). Seedling and post-transplant stages were the best periods to apply BC79 for control of rice blast. The B. methylotrophicus BC79 strain hence has enormous potential as an agricultural agent for biocontrol of rice blast.     

Transgene Stacking and Coordinated Expression of Plant Defensins Confer Fungal Resistance in Rice

Transgenic rice with cleavable chimeric polyprotein and single-protein gene constructs placed under the control of single maize ubiquitin promoter were generated by Agrobacterium tumefaciens-mediated transformation. The polyprotein precursor consists of a leader peptide and two different antimicrobial proteins (AMPs), Dm-AMP1 and Rs-AFP2, from the seeds of Dahlia merckii and Raphanus sativus, respectively. These genes were linked by a 16 amino acid Ib-AMP linker peptide region isolated from the seeds of Impatiens balsamina. Average expression of Dm-AMP1 and Rs-AFP2 was 8.35?±?0.56 and 8.1?±?0.6 2 g/mg of total soluble proteins in the leaf extracts of transgenic plants. Plants transformed with polyprotein construct showed significantly improved disease resistance against Magnaporthe oryzae and Rhizoctonia solani by 90% and 79%, respectively, as compared to untransformed plants. The plants transformed with polyprotein construct were more resistant to M. oryzae compared to plants transformed with control single-gene constructs of Dm-AMP1 and Rs-AFP2. The chimeric polyprotein was cleaved and present in equimolar concentration in transgenic rice and the individual AMPs were secreted into the extracellular space. The results of in vitro, in planta, and microscopic analyses suggest that chimeric polyprotein expression has the potential to provide broad-spectrum disease resistance in rice.     

Rhizosphere Bacteria for Biocontrol of Bacterial Blight and Growth Promotion of Rice

Several bacterial strains were isolated from different rhizospheres. Among these, strain PDY7 exhibited strong antibacterial activity against the rice bacterial blight (BB) pathogen Xanthomonas oryzae pv. oryzae (Xoo) by the laboratory dual plate assays. The antibacterial property of the strain PDY7 was further investigated for the production of 2,4-diacetylphloroglucinol (DAPG), which amplified a characteristic of 629-bp DNA fragment by PCR-based screening method using phlD primers. The application of phlD positive strains was carefully evaluated for disease control and growth promotion of rice plants under field conditions. The selected strain PDY7 suppressed the rice BB by 58.83% and 51.88% under glass house and field conditions, respectively. In addition, the strain PDY7 showed significant two-fold increase in root length (18.08 cm), shoot length (29.81 cm), and grain yield (96.07 g). Strain PDY7 promoted the growth of rice plants by production of indole-3-acetic acid (IAA), which was determined by high performance liquid chromatography (HPLC) analysis. Our findings suggest that PDY7 belongs to the P. fluorescens group and can serve as potential biocontrol of BB as well as biofertilizer agent for growth promotion of rice.     

Occurrence of Alternaria leaf blight of sunflower caused by two closely related species Alternaria solani and A. tomatophila in Inner Mongolia

Sunflower (Helianthus annuus L.) is an important oilseed crop in China and is grown in rotation with potato in the northern parts of China, including Inner Mongolia, Hebei, Shaanxi, Shanxi, Gansu, Jilin, Liaoning and Heilongjiang provinces. In 2014, diseased samples of sunflower (variety 3638C) with black sunken lesions on bracts and stems of sunflower were collected in Wuchuan, Hohhot (41°05′ N, 111°27′ E) and Chahaeryouyihouqi, Wulanchabu (41°52′ N, 113°0′ E), Inner Mongolia, China. We used morphological and molecular techniques to identify the causal agent of Alternaria leaf blight. Phylogenetic analysis based on a combined phylogenetic dataset using the glyceraldehyde-3-phosphate dehydrogenase (gpd) and Alternaria major allergen gene (Alt a1) was done to support the morphological identification. In addition, PCR was performed with specific primers targeted on Alt a1 and calmodulin genes of A. solani and A. tomatophila. The results showed that HaAs7 gave an amplicon of the expected size (164 bp) with A. solani-specific primers, however, no band was observed with the A. tomatophila-specific primers. The opposite pattern of PCR result (483 bp) was obtained with DNA of HaAt4 as template, indicating that HaAs7 is A. solani and HaAt4 is A. tomatophila. Furthermore, evaluation the pathogenicity of the recovered Alternaria isolates and their potential as causal agents of Alternaria leaf blight of sunflower was done. Pathogenicity tests showed that A. solani and A. tomatophila tested could be capable of causing Alternaria leaf blight of sunflower. Till now, nine species of Alternaria have been reported causing Alternaria leaf blight of sunflower, this is the first report that A. solani and A. tomatophila as causal pathogens for Alternaria leaf blight on sunflower.