Jingao Dong

Natural occurrence of fumonisins B1 and B2 in corn in four provinces of China

Fumonisin B1 (FB1) and fumonisin B2 (FB2) are the most abundant fumonisins (FBs) occurring worldwide in maize, infected mainly by Fusarium verticillioides and F. proliferatum. A total of 307 corn kernel samples were collected from 45 districts of Gansu, Shandong, Ningxia and the Inner Mongolia provinces of the north and northwest China. The samples were analysed for FB1 and FB2 by high-performance liquid chromatography. The FBs (FB1+ FB2) incidence rate in samples from Gansu, Shandong, Ningxia and Inner Mongolia were 31.5%, 81.1%, 46.2% and 53.6%, respectively. Average FBs concentration was 703 μg/kg and the concentrations ranged from ≤11 to 13,110 μg kg−1. Results were compared with the European Commission (EC) regulation for FB1+ FB2 in unprocessed maize for human consumption of 4 mg kg−1. Contamination in 17 samples was higher than these levels. More than 80% of the samples from Liaocheng county, which is located in the Shandong province, were contaminated with FBs, with a mean total FB concentration of 2496 μg/kg. The result was significantly different from that of the Inner Mongolia (1399 μg/kg), Ningxia (373 μg/kg) and Gansu (175 μg/kg). Average exposure to FBs (0.12 μg/kg body weight/day) is within the provisional maximum tolerable daily intake of 2.0 mg/kg of body weight set by the Joint Food and Agriculture Organization and World Health Organization Expert Committee on Food Additives.

Natural incidence of Fusarium species and fumonisins B1 and B2 associated with maize kernels from nine provinces in China in 2012

Fusarium species, which can produce mycotoxins, are the predominant pathogens causing maize ear rot, a disease that results in severe economic losses and serves as a potential health risk for humans and animals. A survey was conducted in 2012 to investigate the contamination of maize by Fusarium species and fumonisins B1 and B2. A total of 250 maize samples were randomly collected from nine provinces (Hebei, Shanxi, Inner Mongolia, Yunnan, Sichuan, Guizhou, Heilongjiang, Liaoning and Ningxia) in China. Fusarium species were isolated and identified using morphological (electron microscope) and molecular methods (polymerase chain reaction (PCR) and sequencing). Fumonisins B1 and B2 were analysed using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) with OPA (2-Mercaptoethanol, o-phthaldialdehyde) post-column derivatisation. A total of 2321 Fusarium isolates (20.7%) were obtained from all the samples. These isolates included nine Fusarium species, namely, F. graminearum, F. verticillioides, F. subglutinans, F. proliferatum, F. temperatum, F. oxysporum, F. equiseti, F. meridionale and F. chlamydosporum. The incidence of occurrence of Fusarium species in Guizhou was the highest, while in Inner Mongolia it was the lowest. F. verticillioides was the dominant species of maize ear rot in Liaoning, Sichuan, Hebei and Ningxia. F. graminearum was the dominant species in Yunnan, Guizhou and Shanxi. F. subglutinans was the dominant species in Heilongjiang. F. verticillioides and F. graminearum percentages were the same in Inner Mongolia. The incidence of fumonisins in Liaoning was high (up to 81.0%) and in Heilongjiang low (up to 10.3%). Except Shanxi, more than 50% of maize samples from other provinces were contaminated with fumonisins, with concentrations less than 500 ng g−1. About 33% of maize samples from Yunnan were contaminated with high levels of fumonisins, and average of fumonisin levels were 5191 ng g−1. Fusarium species causing maize ear rot in different areas in China were highly diverse and such areas with exposure to high levels of fumonisin contamination have a potential health risk for human and animals.

Degradation of nicosulfuron by Bacillus subtilis YB1 and Aspergillus niger YF1

The optimal degrading conditions for the nicosulfuron degradation by Bacillus subtilis YB1 and Aspergillus niger YF1, and site of their action on nicosulfuron were studied. The results showed that the degradation efficiency of free cells of B. subtilis YB1 and A. niger YF1 was respectively 87.9 and 98.8% in basic medium III containing 2 mg/l of nicosulfuron after inoculation with 1 ml of culture containing 2.3 × 107 CFU ml−1 and incubation for 5 days at 35°C. Moreover, the degradation rate of nicosulfuron by the mixture of microorganisms was much higher than for every of them taken separately in the same conditions. The mass spectrometric analysis of the products degraded by B. subtilis YB1 revealed that the sulfonylurea bridge in nicosulfuron molecule had been broken. Extracellular (EXF) and endocellular (ENF) fractions obtained from bacterium and fungus were tested for the ability to degrade nicosulfuron. The degradation efficiency of fractions extracted from B. subtilis YB1 was 66.8% by EXF and 15.8% by ENF, but neither EXF nor ENF extracted from A. niger YF1 had the activity of degrading nicosulfuron.

Fumonisins B1 and B2 in maize harvested in Hebei province, China, during 2011–2013

A total of 125 maize kernel samples were collected from Hebei province in China during 2011–2013 and were analysed for incidence and contamination levels of fumonisins (FB1 + FB2) by HPLC with fluorescence detection. The incidence of FBs for all samples was 46.4%. The mean contamination level of FBs for the samples collected in 2013 was 706 μg kg−1, which was higher than the levels in 2012 (429 μg kg−1) and 2011 (250 μg kg−1). All samples, except five, exhibited total FB levels below 4000 μg kg−1, which is the maximum limit as set by the European Commission. The probable daily intakes of FBs (0.04 in 2011; 0.07 in 2012; 0.12 in 2013, expressed as μg kg−1 body weight/day) were all within the provisional maximum tolerable daily intake of 2.0 μg kg−1 of body weight/day as set by the Joint FAO/WHO Expert Committee on Food Additives. Nevertheless, monitoring is needed to prevent and control the potential risk of FB exposure to the consumers.

StRas2 regulates morphogenesis, conidiation and appressorium development in Setosphaeria turcica.

The proteins of Ras family are a large group of monomeric GTPases and act as molecular switches transducing extracellular signals into the cell in higher eukaryotes. However, little is known about roles of Ras family in the foliar pathogens. In this research, we cloned the gene named StRas2 encoding Ras in Setosphaeria turcica and investigated its function by RNA interference technology. We found that the growth rate of RNAi transformants named as R1, R2, R3, R4, R5 and R6, in which the StRas2 silencing efficiency fell in turn. With the highest silencing efficiency, the transformant R1 showed anomalistic hyphae morphology, indicating its growth was significantly affected. The transformants with a middle-silencing efficiency, such as R3, R4, displayed a delay when forming appressoria and invasive hyphae. R1 could not form conidia and appressoria. However, the conidial formation in R5 and R6 was significantly reduced, and these two transformants could form appressoria and penetrate the artificial cellophane, only that its invasive hyphae were fascicular and rarely branched. The HT-toxin biological activity of all transformants showed no difference. All results suggested that StRas2 is involved in the morphogenesis, conidiation, and appressorium development and is not related to the biosynthesis of HT-toxin.

The Extraction, Isolation and Identification of Exudates from the Roots of Flaveria bidentis

Large amounts of ”Flaveria bidentiss” root culturing solution were obtained by using DFT (deep flow technique) equipment and these solution which was vacuum concentrated (10, 20 mg mL^(-1)) can have a certain inhibition on ”Triticum aestivum”, ”Cucumis sativus”, ”Raphanus sativus”, ”Amaranthus retroflexus”, ”Setaria viridis”, ”Chenopodium album”, ”Echinochloa crusgalli” and ”Chloris virgata”. This outcome suggested some active compounds in the root exudates of ”Flaveria bidentis” can inhibit the germination, seedling elongation and root length. The dichloromethane extract of root exudates was identificated by GC-MS, and 29 kinds of compounds, including esters, hydrocarbons, ketones, thiazole, amines, etc. were obtained and the phthalate n-octyl ester, phthalate 2-ethylhexyl ester were proved to be allelochemicals. The culturing solution of root exudates was separated through the resin column and silica gel column and a component inhibiting seedling height, root length and fresh weight of wheat was got. There were 6 kinds of organic compounds in this component including dioctyl phthalate, 1,2-phthalate, mono(2-ethylhexyl) ester by GC-MS.

StSTE12 is required for the pathogenicity of Setosphaeria turcica by regulating appressorium development and penetration.

In filamentous fungi, the pathogenic mitogen-activated protein kinase (PMK) pathway performs an important function in plant infection. STE12-like genes found in higher eukaryotes encode transcription factors and are regulated by the PMK pathway. However, the functions of STE12-like genes in foliar pathogens remain poorly understood. In this study, we cloned StSTE12 from Setosphaeria turcica and investigated its functions by RNA interference. Transformants ste12-3, ste12-2 and, ste12-1, in which the StSTE12 silencing efficiency increased in order, were confirmed by real time PCR. Compared with the wild-type (WT) strain, the transformants showed reduced growth rate, lighter colony color, and obviously decreased conidium production. More importantly, different to WT strain and ste12-3 with lower StSTE12silencing efficiency, ste12-1 and ste12-2 with higher StSTE12 silencing efficiency were nonpathogenic on intact leaves, but pathogenic on wounded leaves. However, the biological activity of HT-toxin from all transformants showed no difference on corn leaves. Furthermore, ste12-1 and ste12-2 did not penetrate artificial cellophane membrane and showed abnormal and delayed development appressoria. Although it could penetrate the cellophane membranes, ste12-3 formed appressoria after 48 h of inoculation more than WT. Therefore, StSTE12 was involved in vegetative growth, conidiation, appressorial development, penetration as well as the pathogenicity, but it was not related to HT-toxin biosynthesis. More interestingly, all the results suggested that StSTE12 was crucial for pathogenicity due to involvement in regulating appressoria development and penetration.

Isolation, Identification, and Herbicidal Activity of Metabolites Produced by Pseudomonas aeruginosa CB-4

Abstract CB-4, a bacterial strain with highly effective herbicidal activity, was isolated from infected corn leaves. Through morphology, physiological and biochemical tests, and 16S ribosomal DNA gene sequencing methods, CB-4 was identified as Pseudomonas aeruginosa . We conducted activity-evaluation experiments in the laboratory to assess the herbicidal potential of metabolites produced by strain CB-4. Crude extracts of strain CB-4 have high inhibition activity on Digitaria sanguinalis. In general, the root and shoot growth parameters of D. sanguinalis were significantly reduced by metabolites of strain CB-4. The IC 50 of the culture filtrate extracts for the radicula and coleoptile of D. sanguinalis were 0.299 and 0.210 mg mL −1 , respectively. Component 2 of the herbicidal activity of the crude toxin from strain CB-4 was successfully purified for the first time by using high-speed counter current chromatography with a two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-water (4:5:4:5, v/v) and high-performance liquid chromatography. We concluded that the metabolites of strain CB-4 have the potential to be developed as a microbe-based herbicide.

Purification and Structural Analysis of a Selective Toxin Fraction Produced by the Plant Pathogen Setosphaeria turcica

Thirteen fractions from the pathogenic plant fungus Setosphaeria turcica race 1 were separated and collected using high performance liquid chromatography (HPLC). Their toxic activities were assayed through leaf puncturing on corn differentials (OH43, OH43Ht1, OH43Ht2, and OH43HtN), and the results revealed that eight fractions were toxic and fraction 6 was specifically toxic to OH43Ht1, which could be taken as a gene-selective toxin fraction. Fraction 6 was finely purified via HPLC and condensed by freeze desiccation. Its chemical structure was analyzed with EI-MS, IR, HMBC, 1H-NMR, and two-dimensional NMR. The results suggested that fraction 6 contained an unsaturated double bond, carbonyl and methylene groups with molecular weight of 142.

Isolation and Structural Speculation of Herbicide-Active Compounds from the Metabolites of Pythium aphanidermatum

Natural herbicides, or environment-friendly bioherbicides have been attracted more and more attentions. Isolation and structural identification of natural herbicide-active compounds from plant pathogens has been proved to be an effective approach for novel lead discovery of the pesticide development. In this study, the metabolites of the mutant strain PAM1, which obtained from PA1 of Pythium aphanidermatum (Eds.) Fitzp by ultraviolet radiation were separated and identified by HPLC, NMR, and IR. The results revealed that three active compounds including 4-hydroxy-3-methoxycinnamic acid and two indole derivatives, exhibited inhibition activity on the elongation of radical and coleoptile of Digtaria sanguinalis (L.) Scop.