Inoculation with chlamydospores of Trichoderma asperellum SM-12F1 accelerated arsenic volatilization and influenced arsenic availability in soils

Fungi capable of arsenic（As） accumulation and volatilization are hoped to tackle As-contaminated environment in the future. However, little data is available regarding their performances in field soils. In this study, the chlamydospores of Trichoderma asperellum SM-12F1 capable of As resistance, accumulation, and volatilization were inoculated into As-contaminated Chenzhou（CZ） and Shimen（SM） soils, and subsequently As volatilization and availability were assessed. The results indicated that T. asperellum SM-12F1 could reproduce well in As-contaminated soils. After cultivated for 42 days, the colony forming units（cfu） of T. asperellum SM-12F1 in CZ and SM soils reached 10^10–10^11 cfu g^–1 fresh soil when inoculated at a rate of 5.0%. Inoculation with chlamydospores of T. asperellum SM-12F1 could significantly accelerate As volatilization from soils. The contents of volatilized As from CZ and SM soils after being inoculated with chlamydospores at a rate of 5.0% for 42 days were 2.0 and 0.6 μg kg^–1, respectively, which were about 27.5 and 2.5 times higher than their corresponding controls of no inoculation（CZ, 0.1 μg kg^–1; SM, 0.3 μg kg^–1）. Furthermore, the available As content in SM soils was decreased by 23.7%, and that in CZ soils increased by 3.3% compared with their corresponding controls. Further studies showed that soil p H values significantly decreased as a function of cultivation time or the inoculation level of chlamydospores. The p H values in CZ and SM soils after being inoculated with 5.0% of chlamydospores for 42 days were 6.04 and 6.02, respectively, which were lowered by 0.34 and 1.21 compared with their corresponding controls（CZ, 6.38; SM, 7.23）. The changes in soil p H and As-binding fractions after inoculation might be responsible for the changes in As availability. These observations could shed light on the future remediation of As-contaminated soils using fungi.

Studies on the Correlation between Germination and cAMP Content of Different Color Chlamydospores in Ustiloginoidea virens

Ustilaginoidea virens is the causal agent of a serious disease of rice. To reveal the relationship between germination and the 3'-5'-cyclic adenosine monophosphate(c AMP) content from the dormant(black or green-black) and non-dormant(yellow)chlamydospore in Ustiloginoidea virens,this study adopts ultrasonic-bath method and high-performance liquid chromatography(HPLC) method, for extraction c AMP content of the different color chlamydospore. The results demonstrated that, as for the freshly chlamydospores collected from the false smut balls during their germination, c AMP content of yellow chlamydospore appeared a slight growth during 0-12 h, and showed a rapid declining as the germination time extended(12-48 h). Above all, in yellow chlamydospores, the germination rate and the content of c AMP presented a very notably negative correlation(|r|=0.861 9>r0.01=0.834), but the correlation between germination rate and content of c AMP was not obvious in black chlamydospores. The germination rate and the content of c AMP presented a notably positive correlation under different storage period(0, 2, 4, 6, 8,10 months) of the two color chlamydospores(the yellow of chlamydospore: |r | = 0.785 1 > r0.05= 0.707; the black of chlamydospore: |r| = 0.957 9 > r0.05= 0.707).

Inhibition of Chlamydospore Germination and Mycelial Growth of Trichoderma spp. by Chemical Fungicides

For exploring the influences of application and residue of chemical fungi- cides on chlamydospore preparations of Trichoderma spp., the effects of seven chemical fungicides on chlamydospore germination and mycelia growth of two bio- control fungi T. harzianum 610 and T. Iongibrachiatum 758 were studied. Carben- dazim, tebuconazole and difenoconazole showed strong toxicities, thiram and car- bexin showed moderate toxicities, and metalaxyl showed Mycelia growth of the two strains was more sensitive to most tested fungicides than those of chlamydospore germination. Chlamydospore germination of 610 was more sensitive to tested fungicides than those of 758, and mycelia growth of 758 was more sensitive to most tested fungi- cides than those of 610. Among the seven fungicides, 98% carbendazim had the strongest effects （ECru values were 1.64 and 0.05μpg/ml）, and 70% pentachloroni- trobenzene had the weakest effects （EC50 values were 1.64 and 0,05 μg/ml） for chlamydospore germination and mycelia growth of 610. As for 758, 98% carbendaz- im had the strongest inhibitory effects and 95% metalaxyl had the weakest inhibitory for chlamydospore germination of 756 （EC50 values were 0.62 and 1 108.61 μg/ml respectively）, whereas 96.2% tebuconazole showed the strongest inhibitory effects for mycelia growth of 758 （EC= value was 0.32μg/ml）, and 95% metalaxyl was the weakest （EC= value was 1 206.29 μg/ml）. According to the applied concentration of different fungicides in practice, we concluded that chlamydospore preparations of 610 and 758 could not be combined with carbendazim, tebuconazole, thiram and carboxin for controlling plant diseases, and the pesticide residues to the biocontrol effects should be kept in mind. Chlamydospore preparations of 610 and 758 can be and difenoconazole for controlling plant dis- eases, 758 chlamydospore preparations and germinated chlamydospore of 610 can be combined with metalaxyl for controlling plant diseases, and pesticide residue risk was not serious.

<i>Ustilaginoidea virens</i>Infection of Rice in Arkansas: Toxicity of False Smut Galls, Their Extracts and the Ustiloxin Fraction

Cool, wet conditions in the southern US during the maturing stages of rice in 1998 contributed to outbreaks of false smut caused by Ustilaginoidea virens. Water extracts of false smut galls in Asia have been reported to contain ustiloxin toxins, cyclic peptide antibiotics that interfered with microtubule function and caused “lupinosis”-like lesions in mice. Cell-free extracts from false smut galls on rice grown in Arkansas were fractionated by a published procedure for the purification of ustiloxins. The ustiloxin fraction was phytotoxic to Lemnapausicostata (duckweed) at ≥19 μg/ml, but the host plant, rice, was much less susceptible, exhibiting phytotoxic effects in germinating seeds at ≥1000 μg/ml. The aqueous extract of rice false smut galls showed no cytotoxicity to mammalian cell cultures at 200 μg/ml, but the ustiloxin fractionwas cytotoxic at 10 - 100 μg/ml. However, rice false smut galls were not toxic when fed to mice at 10% of chow, but caused feed refusal at higher concentrations. We conclude that for 1) the U. virens which causes false smut in southern USA differs from Asian isolates in that does not produce detectable ustiloxins;and 2) false smut affects the appearance, but not the food safety of rice in the United States.

Statistical culture-based strategies to enhance chlamydospore production by Trichoderma harzianum SH2303 in liquid fermentation

Trichoderma-based formulations are applied as commercial biocontrol agents for soil-borne plant path- ogens. Chlamydospores are active propagules in Trichoderma spp., but their production is currently limited due to a lack of optimal liquid fermentation technology. In this study, we explored response surface methodologies for opti- mizing fermentation technology in Trichoderma SH2303. Our initial studies, using the Plackett-Burman design, iden- tified cornmeal, glycerol, and initial pH levels as the most significant factors （P〈0.05） for enhancing the production of chlamydospores. Subsequently, we applied the Box-Behnken design to study the interactions between, and optimal levels of, a number of factors in chlamydospore production. These statistically predicted results indicated that the highest number of chlamydospores （3.6×108 spores/ml） would be obtained under the following condition： corn flour 62.86 g/L, glycerol 7.54 ml/L, pH 4.17, and 6-d incubation in liquid fermentation. We validated these predicted values via three repeated experiments using the optimal culture and achieved maximum chlamydospores of 4.5×108 spores/ml, which approximately a 8-fold increase in the number of chlamydospores produced by T. harzianurn SH2303 compared with that before optimization. These optimized values could help make chlamydospore production cost-efficient in the future development of novel biocontrol agents.