Multi Key Techniques For The Commercialization Of Alternaria Alternata AAC-Toxin Bioherbicide

Alternaria alternata （Fr.） Keissler is a natural pathogen to crofton weed （Eupatorium adenophorum）, one of the malignant weeds in the world. The phytotoxin produced by the fungus is the main factor that can cause the disease on crofton weed and other weeds in arable land. During the earlier period, mass-production, the extraction craft procedure, isolation, identification, bioactivity, action mechanism and specificity of the AAC-toxin had been studied, which exhibits the development potential of the toxin for bioherbicide. Then tenuazonic acid was identified as the main toxic compound in AAC-toxin. However, above study results are not still satifatied with the requirements of basic data for commercialization of this product. On the basis of these results, in this study the extraction craft procedure was improved, and the further evaluation on the development potential of AAC-toxin for bioherbicide was conducted. The herbicidal activity, toxicology, degration in soil and some other characteristics of tenuazonic acid were also studied. The purpose of this studey is to provide essential data for commercialization and evaluate further potential for developing the toxin into bioherbicide.The main contents are given as follows:1. Improvement of extraction craft procedure, herbicidal activity, and emulsifiable concentrates formulation of AAC-toxinThe adsorbability for different macroporous resin DA201, D101, HZ-803, 1300 and activated carbon to Alternaria alternata AAC-toxin was compared. The results showed that DA201 had the best adsorbability, with highest adsorption rate of 72.88% through bioassay of activity of the filtrate after being adsorbed by DA201 and its desorbed filtrate. DA201 was suitable for purification and enrichment of the toxin through column chromatography. Furthermore, optimum adsorption and desorption conditions for DA201 were made as follows: the loaded amount was in culture filtrate/resin of 60-70/1 （v/v）, pH was 4.12-4.62, and 90% ethanol was used for eluting the toxin.Phytotoxicity of AAC-toxin on the main weeds in arable land, crops and Digitaria sanguinalis with different leaf ages was bioassayed. The efficacy against Digitaria sanguinalis and security to wheat and cotton of the toxin were investigated in fields. The results in seedling spraying assay showed that the toxin could efficiently control the main weeds and Digitaria sanguinalis before three-leaf age, and was safe to cotton. At the concentration of 4000/zg/mL, the efficacy of the toxin against 12 weeds was higher than 90%. The weeds were as follows: Trifolium repense, Digitaria sanguinalis, Cyperus difformis, Eclipta prostrata, Echinochloa crusgalli, Geranium carolinianum, Commelina communis, Sonchus asper, Vicia sativa, Ambrosia artemisiifolia, Conyza bonarinsis, Acalypha australis. The injury rate and fresh weight inhibition rate of cotton plants were only 2.96% and 8.67% respectively in field trial. The results in field assay were consistent with that in greenhouse assay. These results suggested that the crude toxin could be used as bioherbicide in cotton field.The surface tension, dispersion, stability and bioactivity of seven emulsifiable concentrates were tested. The results showed that emulsifiable concentrates A, C, D, F and G had nice dispersion, A, C, D and G had nice stability, D and F had high bioactivity, but A and G had larger surface tension. In general consideration, emulsifiable concentrate D, in which triton X-100 was used as emulsifier had higher potential for development.2. HPLC detection, herbicidal activity, toxicity and degradation in soil of tcnuazonic acidHPLC method was established for analysis of tenuazonic acid using a C18 re-phase chromatography column, acetonitrile/methanol/water （5:3:2, by vol） as the mobile phase, and UV detection at 248 nm. The standard deviation was 0.53, coefficient of variation was 1.14%, recovery was 97.17%～100.56%, and the liner correlation cocfficicnt was 0.996. Tcnuazonic acid production ability of 19 Alternaria alternata isolates was compared using combination of the above HPLC analysis method with bioassay of leaf puncture assay. The results showed that among 19 isolates, X501 had the highest productivity of tenuazonic acid with its concentration of 3.8400/zg/mL in filtrate, followed by NEW. The isolates 501-2, 501-3 and YN had weaker pathogenicity.The effects of 11 adjuvants including cmulgcnt 6201 on herbicidal activity of tcnuazonic acid wcrc studied by means of detached leaf puncture assay. The results showed that sodium dodccyl benzene sulfonate had the best enhancement effect, and triton X-100, CGN-3, synergist YZ905 and EF8108-Ⅱexhibited enhancement effect too. The optimum concentrations of the five adjuvants ranged from 1‰～5‰for enhancement effects. Two formulations wcrc designed, formulationⅠis worthy of more development study.Detached leaf puncture assay were used to evaluate the bioactivity of tenuazonic acid to Eupatorium adenophorum from 6.25μg/mL to 400μg/mL. The results suggested that tenuazonic acid was very pathogenitic to Eupatorium adenophorum. Pathogenicity of tenuazonic acid of 50/zg/mL to the leaves at different parts of Eupatorium adenophorum ZG population plant was also investigated using detached leaf puncture assay. The results showed that tenuazonic acid was more pathogenitic to mature leaves than to young ones.About the weed control spectrum and crop security of tenuazonic acid, the results in detached leaf puncture assay showed that, tenuazonic acid was very pathogenetic to 26 weeds among 39 assayed weeds but safe to cotton and tobacco at the concentration of 50μg/mL. At the concentration of 62.5～1000μ^g/mL in seedling spraying assay, tenuazonic acid was all pathogenetic with different degrees to 15 weeds but also safe to cotton and tobacco. These results indicated that tenuazonic acid can be developed as a bioherbicide for weed control in cotton and tobacco fields.The toxicity of tenuazonic acid on Chlamydomonas reinhardtii was studied. The results revealed that tenuazonic acid promoted the growth of C. reinhardtii at low concentrations ranging from 12.5 to 25/zg/mL, however, with increase of tenuazonic acid concentrations, marked inhibition of the growth and impact of chl contents were observed. The 96h EC₅₀（G） and EC_50（Chl） of tenuazonic acid to C. reinhardtii were 310.36μg/mL and 294.27μg/mL respectively. According to the toxicant classification standard with 3μg/mL, the EC₅₀ values of tenuazonic acid were very low for aquatic algae, indicating it may be rather safe to the aquatic ecosystem.The effects of tenuazonic acid on micronucleus and karyokinesis of Vicia faba root tip cells were investigated to evaluate its cytogenetic toxicity. The results indicated that when its concentration was 12.5～50μg/mL, no significant effects of tenuazonic acid on micronucleus ratio and karyokinesis index were found compared with control. However, as the concentration was been increased to 100～400μg/mL, the micronucleus ratio significantly increased whereas karyokinesis index significantly decreased. Multi-nucleolus was the phenomen of abnormal karyokinesis of Vicia faba root tip cells induced by tenuazonic acid, and multi-nucleolus ratio was significantly increased at the concentration ranging from 200 to 400/zg/mL.The cytotoxicity of tenuazonic acid on 3T3 mouse fibroblasts, Chinese hamster lung cells, L-O2 human hepatocytes was investigated by MTT method. The results suggested that, at the concentrations ranging from 12.5 to 400/zg/mL, tenuazonic acid inhibited the proliferation of the three cell lines. The inhibition was enhanced with the increase of tenuazonic acid concentrations and exposure time. Tenuazonic acid was more cytotoxic to 3T3 and CHL than L-02, and 3T3 was the most susceptible to it. The ECso values for them to tenuazonic acid at 24 h exposure were 41.64μg/mL, 59.33μg/mL and 85.98μg/mL respectively.Degradation of tenuazonic acid in soil was investigated under laboratory and field conditions. The results showed that, under laboratory condition, the soil microorganisms obviously accelerated the degradation of tenuazonic acid, suggesting that microorganisms play an important role in degradation of tenuazonic acid. Sunlight also involved in degradation of tenuazonic acid, but was not as important as microorganisms do. In addition, the increase of water contents and temperature of soil promoted the degradation of tenuazonic acid in soil. The studies under field condition revealed that the half life of tenuazonic acid was only about 3.22 days, and the residual period was about 20 days in soil.These results above showed that tenuazonic acid has broad weed control spectrum but is safe to cotton and tobacco. And it is environmentally benign, lowly mutagenic, and quickly degradable. All these characteristics of tenuazonic acid suggest its potential for developing into a bio-basedherbicide.