Etiology, Control And Phytotoxin Activity Of Garlic Leaf Blight Caused By Stemphylium Solani

Leaf blight of garlic is a destructive disease in Hubei province, China. Symptoms were observed on infected leaves in Dangyang County from autumn 2005 to spring 2009, with the diseased area estimated to be over 7000 ha. Garlic yield was reduced by 20% on average with up to 70% yield losses in some fields.Leaf blight of garlic has become the main disease limiting garlic production and quality. In primary work, the causal agent of garlic leaf blight was identified as Stemphylium solani from cultural and morphological characteristics, and subsequent analysis of the internal transcribed spacer region of ribosomal DNA.And then, aetiology, epidemiology and integrated control of garlic leaf blight caused by S. solani, and pathogenic mechanism of phytotoxin produced by the pathogen were also studied in this paper. The main research results were listed as follows:1.Diseased leaves with leaf blight symptoms were collected from fields in the main garlic production areas of Dangyang County in Hubei province, China. Thirty-one Stemphylium sp.isolates and 33 Alternaria sp. isolates were obtained using usual tissue isolation method. The Stemphylium sp. isolates DY-1,DY-5 and DY-6, and Alternaria sp. isolates DY-P-1 and DY-W-1 were used for pathogenicity test. By inoculation with fungal plugs or conidial suspension, white spots were observed on inoculated leaves for isolates DY-1,DY-5 and DY-6, but no symptoms were seen on garlic plants treated with isolates DY-P-1 and DY-W-1.The isolates DY-1,DY-5 and DY-6 were transferred to PSA plates. On PSA, conidiophores were up to 169μm long. Conidia were pointed at the swollen apex of each conidiophore, brown, with one to three dark coloured transverse septa and distinctly constricted at median septa, two to seven longitudinal or oblique septa, 29-58(45)×14-28(22)μm (mean) and mean length/width ratio was 2.0.On naturally diseased leaves of garlic, the size of conidia were ranged from 27-48(39)×15-30(21)μm. Based on cultural characteristics and fungal morphology of Simmons and Ellis, the isolates were identified as S. solani. Genomic DNA was extracted from three isolates, and sequences of rDNA-ITS were obtained. Comparison with sequences in GenBank showed 99% similarity with S. solani. By neighbor-joining method, the phylogenetic tree of the ITS1-5.8S-ITS2 sequences from Stemphylium species was constructed. Stemphylium solani (AF203449,AF203450), S. nabarii (AY372679), S. subglobuliferum (AY751454) and isolates DY-1,DY-5 and DY-6 were grouped together.2.Biological characteristics of S. solani isolate DY-5 were tested by measuring mycelial growth in different conditions.For the growth of mycelia on PSA medium, the range of temperature was 5-35℃,with the optimum of 20-30℃.The favorable pH value was 4-10, with the optimum pH 6-8.Among the tested carbon and nitrogen sources in Czapek liquid medium, mannitol and urea were not good for the growth of mycelia. Starch and glutamate were the most favorable for its growth. The lethal temperature for the mycelium was 55℃for 10 minutes.The infection process of S. solani on susceptible garlic leaves was studied by light microscopy technique. The infection process was investigated by light microscopy from 2 to 120 h after inoculation (AI).Spore germination was observed within 2 h AI and up to 11 germtubes formed within 6 h AI. The pathogen invaded the garlic leaf only through stomates. Infection hyphae were observed inside stomates within 8 h AI on garlic leaves, and more stomates were infected by secondary hyphae within 12 h AI. Abundant hyphae were produced within 24 h AI and more frequent infection was observed within 24,36,48,72,96 and 120h after inoculation.3.To determine the property of the toxins, the culture filtrates were treated in various ways. No toxin could be detected in the precipitates after adding methanol or acetone. After partitioning with normal butanol or ethyl acetate, the most toxins were detected in solvent fractions.The toxic activity in culture filtrates was stable even after incubation at 121℃for up to 30 min. Culture filtrates treated with either both proteinase K or pepsin was still toxic to garlic leaves.Filtrates incubated at pH 2 to 4 for 24 hr and then re-adjusted to pH 7 showed the most toxic activity in the leaf necrosis assay, while the toxic activity decreased with increasing pH from 5 to 13.Pathogenicity of S. solani to various crops and garlic cultivars, and toxicity of its culture filtrates indicated that toxin(s) produced by S. solani were non-host-specific. 4.A non-host-specific phytotoxin from culture filtrate of S. solani was isolated by using TLC and HPLC. Preparative analytical TLC plates spotted with toxin samples were developed separately in 24 solvent systems. The most bands (total 13 fractions) from the toxin on TLC plates was obtained with ethyl acetate/sherwood oil/methanol (4:1:0.35, vol/vol/vol), and this optimal solvent system was used for preparative TLC.Only the fraction with the highest toxicity, the fourth fraction, was collected. This fourth fraction was then purified further with preparative HPLC, and the purified phytotoxin, named SS-toxin(100 mg), appeared as red-brown crystals. Based on chemical identification, the reaction was positive in ferric chloride test. The SS-toxin exhibited UV absorption maxima at 215 run,264 nm and 458 nm, and this indicated an aromatic structure for the toxin. The IR spectrum displayed characteristic absorptions for hydroxyl groups (3400 cm-1),methyl groups (2946 cm-1,1449 cm-1 and 1395 cm-1),aromatic rings (3092 cm-1 and 1595 cm-1),unchelated CO (1670 cm-1) and conjugated CO (1640 cm-1).The result of ESI-MS together with elemental analysis suggested the molecular formula of C16H16O8 (Mw:336). The structure of SS-toxin was further identified as 7-methoxy-2-methyl-1,2,3,4-tetrahydro-1,2,3,4,5-pentahydroxyanthraquinone or its derivate with 1H-NMR spectrum.5.The physiological assay, transmission electron microscopy and bioassay of standard redox system in the plasma membrane were used to study pathogenic mechanism of SS-toxin. Regression lines for susceptible and resistant cultivars are, respectively, as follows:Y=0.0871X+3.389, R2=0.9774,P<0.05,and Y=0.0546X+ 0.2966, R2=0.9694, P<0.05.The root growth of susceptible cv. Changbanpo was more susceptible to SS-toxin than the shoot growth,and the EC50 values were 64.9 and 178.5μg ml-1,respectively. The numbers of actively dividing cells root tips were also decreased. Chlorophyll content reduction by the SS-toxin detected at all tested concentrations in susceptible cv. Changbanpo, indicated that the symptoms of garlic leaf blight is related with the toxin produced by pathogen. In leaf cells of the susceptible cultivar, the earliest toxin-induced ultrastructural changes were detected 2 h after toxin treatment in the plasma membranes and cell walls, and these modifications became more severe and more frequent up to 24 h of toxin exposure. The number of mitochondrial cristae was reduced in leaves of the susceptible cultivar by 6 h after treatment with the toxin. Disordered chloroplast lamellae, swollen chloroplasts and disrupted nuclear membranes appeared after 12 h of exposure to the toxin for the susceptible cultivar. No apparent change of rough endoplasmic reticulum was observed even after 24 h. In leaf cells of the resistant cultivar, the earliest effects (plasma-membrane invagination and cell wall changes) were detected 12 h after toxin treatment. The numbers of mitochondrial cristae were reduced by 24 h. Other organelles including chloroplast, nuclear membrane and rough endoplasmic reticulum were seemingly not affected in leaf cells of the resistant cultivar during the first 24 h of exposure.The plasma membranes prepared by aqueous polymer two-phase partitioning from leaves of susceptible and resistant cultivars, showed no significant contamination by other membrane systems, as confirmed by assays tested in the presence of selective inhibitors. Total H+-ATPase activities were more than 70% inhibited by vanadate in both cultivars.The H+-ATPase activities of plasma membranes, isolated from both cultivars, were inhibited by SS-toxin in a dose-dependent manner. When the concentration of toxin was increased from 0.1up to 200 g ml-1,the relative activity of H+-ATPase in plasma membrane extracts of resistant cultivar decreased from 92.1% to 56.4% and for susceptible cultivar, from 93.4% to 41.4%. The NADH oxidation and Fe(CN)63- reduction rates of plasma membranes from both cultivars were inhibited by SS-toxin at all tested concentrations ranging from 0.1 to 200 g ml-1,and the inhibition appeared to be proportional to the concentration of toxin. Using ferricyanide as an electron receptor, the relative NADH oxidation rate in the resistant or susceptible cultivar was reduced to 45.8% or 43.1%,respectively, after treatment with SS-toxin. When NADH was used as an electron donor, the relative Fe(CN)63" reduction rate in the resistant or the susceptible cultivar was decreased to 45.4% and 18.9%, respectively. Our results suggest that, under in vitro condition, the plasma membrane ATPase and standard redox system can be both the cellular targets of SS-toxin.6.Epidemiology, cultivar resistance, and chemical controls of garlic leaf blight were investigated throughout the 2006 to 2008 growing seasons in Dangyang County, Hubei province to improve to disease control methods. In 2006/2007, the initial symptoms of small white spots were observed on 5 November, and a high disease severity was also recorded at final harvest. The months of November, December and March had the highest rates of disease increase, with 37%,14% and 13%,respectively. In 2007/2008, the occurrence of leaf blight was delayed until 24 November, and leaf blight increased very slowly with DSI reaching only a maximum of 27.5 by final harvest. For all treatments (glasshouse, cool storage, in the field on the soil surface, buried in soil at 10 cm or buried at 20 cm), S. solani was consistently recovered from diseased plant debris 165 days after treatment while viable conidia of S. solani were detected only in debris kept in the glasshouse(10.8%) or under cool storage (5.7%). The warm condition and soil surface were suitable for survival of the pathogen. There were significant differences in blight severity between garlic cultivars in the field and cultivars Qingganruanye, Ruanruanye and Zixuan-2 were among the most resistant. For, fungicidal seed treatments, fludioxonil (0.05 a.i. g kg-1) and thiram (1.25 g a.i. kg-1) had significant efficacy. Inhibition of mycelial growth of S. solani by nine fungicides was assayed on amended PSA medium. Flusilazole EC (40%) had the highest inhibitory effect with the 50% effective concentration (EC50) at 0.4μg ml-1.The next most inhibitory were 30% difenoconazole/propiconazole EC,50% carbendazim WP,10% difenoconazole WG and 20.67% flusilazole/famoxadone EC with EC50 values of 1.0,1.4,2.0,2.7μg ml-1, respectively. Fungicidal applications in the field were effective in controlling leaf blight, and 40% flusilazole EC,20.67% flusilazole/famoxadone EC or 75% mancozeb WP had the highest efficacy in reducing leaf blight severity.