Distinct laccase expression and activity profiles of Trametes versicolor facilitate degradation of benzo[a]pyrene.

A Trametes versicolor isolate from the Changbai Mountain showed promising activity in degrading benzo[a]pyrene (BaP), which is a high molecular weight (HMW) polycyclic aromatic hydrocarbon (PAH) compound. It was hypothesized that the T. versicolor isolate encode BaP-degrading enzymes, among which laccase is mostly sought after due to significant commercial potential. Genome of the T. versicolor isolate was sequenced and assembled, and seven laccase homologues were identified (TvLac1-7) as candidate genes potentially contributing to BaP degradation. In order to further identify the BaP responsive laccases, time-course transcriptomic and proteomic analyses were conducted in parallel on the T. versicolor isolate upon BaP treatment. Homologous laccases showed distinct expression patterns. Most strikingly, TvLac5 was rapidly induced in the secreted proteomes (secretomes), while TvLac2 was repressed. Recombinant laccase expression and biochemical characterization further showed corresponding enzymatic activity profiles, where TvLac5 was 21-fold more effective in BaP degradation compared to TvLac2. Moreover, TvLac5 also showed 3.6-fold higher BaP degrading activity compared to a commercial laccase product of T. versicolor origin. Therefore, TvLac5 was concluded to be a BaP-responsive enzyme from T. versicolor showing effective BaP degradation activity.

Genetic Differentiation and Clonal Expansion of Chinese Botrytis cinerea Populations from Tomato and Other Crops in China.

Botrytis cinerea is an important pathogen of vegetable and fruit crops but little is known about its population structure and genetics in China. We hypothesized that the geographic populations of B. cinerea in China would be genetically differentiated by host, geographic location, and/or year. In this study, we collected 393 B. cinerea isolates representing 28 populations from tomato, cherry, and nectarine from 2006 to 2014 in China. The isolates were analyzed using 14 microsatellite markers, including six new markers that provided more genotyping power than the eight previously published loci. We also investigated the B. cinerea population structure and inferred its mode of reproduction and dispersal based on genotype data. High genotypic diversity was detected in all populations, and clonal reproduction was dominant. Southern China populations harbored more genotypes than northern populations. Differentiation by host plant was evident. Between 2011 and 2012, genotypes changed only slightly among years for Liaoning populations, but they changed substantially among years for the Shanghai and Fujian populations. Clonal dispersal was detected and the farthest dispersal distance was estimated to be about 1,717 km. Two high-frequency genotypes were widely distributed in more than 10 populations and across several years. Our results provide useful, novel information for plant breeding programs and control of B. cinerea in China.

Penicillium section Lanata-divaricata from acidic soil.

Penicillium species in section Lanata-divaricata are common soil-inhabiting fungi, but their presence in acidic soil has rarely been investigated. In an ongoing survey of Penicillium species occurring in China, 465 strains were isolated from soil, and of which 60 belonged to section Lanata-divaricata. The majority of these strains were isolated from acidic soil. The phylogenetic relationship between these 60 isolates and accepted species of section Lanata-divaricata was studied using ITS, BenA, CaM and RPB2 sequences, which revealed the presence of seven accepted species and 13 novel lineages. Combining phylogenetic data with data generated during macro- and microscopic observations resulted in the description of 13 new species. The growth rate of the new species obtained in this study was determined under acidic, neutral and alkaline conditions (pH 4, 7, 10). With the exception of P. hainanense, which was not able to grow at pH 10, all strains were able to grow at the three examined pH levels. Eleven species (i.e. P. austrosinense, P. flaviroseum, P. globosum, P. griseoflavum, P. hainanense, P. jianfenglingense, P. laevigatum, P. rubriannulatum, P. soliforme, P. spinuliferum, P. yunnanense) grew faster at low pH (pH 4) than at pH 7 or 10, and these species are therefore referred to as acid-preferential. Penicillium viridissimum grew fastest on neutral medium and P. guangxiense grew best at pH 10, and is therefore considered to be acid-tolerant. By isolating strains from a unique environment, combined with targeted isolation using a well-designed protocol, we are able to describe new fungal diversity with specific physiological characteristics.

Assessing the Risk for Resistance and Elucidating the Genetics of Colletotrichum truncatum That Is Only Sensitive to Some DMI Fungicides.

The genus Colletotrichum contains a wide variety of important plant pathogens, and Colletotrichum truncatum is one of the most prevalent species of Colletotrichum on chili in China. Demethylation-inhibitor fungicides (DMIs) are currently registered chemical agents for the management of the anthracnose disease caused by Colletotrichum spp. To assess the risk for DMI resistance development, 112 C. truncatum isolates were collected from infected pepper in 13 regions of China. The sensitivity of C. truncatum isolates to five DMI fungicides was determined based on mycelial growth inhibition assay. C. truncatum was sensitive to prochloraz, epoxiconazole, and difenoconazole, but not to tebuconazole or myclobutanil. Baseline sensitivity using the 112 C. truncatum isolates was established for the first three effective DMIs. Prochloraz, epoxiconazole, and difenoconazole EC50 values were 0.053 ± 0.023, 1.956 ± 0.815, and 1.027 ± 0.644 µg/ml, respectively. Eleven stable DMI-resistant mutants all exhibited lower fitness levels than their wild-type parents, suggesting a low risk of DMI resistance in C. truncatum. By inducing gene expression, CtCYP51 expression increased slightly in the resistant mutants as compared to wild-types when exposed to DMI fungicides and thus contributed at least partially to resistance. Molecular docking with CYP51 structure models was used to explain differential sensitivity of the DMI fungicides in C. truncatum. Our results suggest that the M376L/H373N mutations in CYP51 changed the conformation of DMIs in the binding pocket. These changes prevented the formation of the Fe - N coordinate bond between the heme iron active site and tebuconazole or myclobutanil, and apparently contributed to tebuconazole and myclobutanil insensitivity of C. truncatum.

Genetic differentiation and recombination among geographic populations of the fungal pathogen Colletotrichum truncatum from chili peppers in China.

Colletotrichum truncatum is an extremely important fungal pathogen. It can cause diseases both in humans and in over 460 plant species. However, little is known about its genetic diversity within and among populations. One of the major plant hosts of C. truncatum is pepper, and China is one of the main pepper-producing countries in the world. Here, we propose the hypotheses that geography has a major influence on the relationships among populations of C. truncatum in China and that infections in different populations need to be managed differently. To test these hypotheses, we obtained and analyzed 266 C. truncatum isolates from 13 regions representing the main pepper-growing areas throughout China. The analysis based on nine microsatellite markers identified high intrapopulation genetic diversity, evidence of sexual recombination, and geographic differentiation. The genetic differentiation was positively correlated with geographic distance, with the southern and northern China populations grouped in two distinct clusters. Interestingly, isolates collected from the pepper-breeding center harbored the most private alleles. The results suggest that the geographic populations of C. truncatum on peppers in China are genetically differentiated and should be managed accordingly. Our study also provides a solid foundation from which to further explore the global genetic epidemiology of C. truncatum in both plants and humans.

Loss of heterozygosity drives clonal diversity of Phytophthora capsici in China.

Phytophthora capsici causes significant loss to pepper (Capsicum annum) in China and our goal was to develop single nucleotide polymorphism (SNP) markers for P. capsici and characterize genetic diversity nationwide. Eighteen isolates of P. capsici from locations worldwide were re-sequenced and candidate nuclear and mitochondrial SNPs identified. From 2006 to 2012, 276 isolates of P. capsici were recovered from 136 locations in 27 provinces and genotyped using 45 nuclear and 2 mitochondrial SNPs. There were two main mitochondrial haplotypes and 95 multi-locus genotypes (MLGs) identified. Genetic diversity was geographically structured with a high level of genotypic diversity in the north and on Hainan Island in the south, suggesting outcrossing contributes to diversity in these areas. The remaining areas of China are dominated by four clonal lineages that share mitochondrial haplotypes, are almost exclusively the A1 or A2 mating type and appear to exhibit extensive diversity based on loss of heterozygosity (LOH). Analysis of SNPs directly from infected peppers confirmed LOH in field populations. One clonal lineage is dominant throughout much of the country. The overall implications for long-lived genetically diverse clonal lineages amidst a widely dispersed sexual population are discussed.

Genetically diverse long-lived clonal lineages of Phytophthora capsici from pepper in Gansu, China.

Phytophthora capsici causes significant loss to pepper production in China, and our objective was to investigate the population structure in Gansu province. Between 2007 and 2011, 279 isolates were collected from pepper at 24 locations. Isolates (or subsets) were assessed for simple sequence repeat (SSR) genotype, metalaxyl resistance, mating type, and physiological race using cultivars from the World Vegetable Center (AVRDC) and New Mexico recombinant inbred lines (NMRILs). The A1 and A2 mating types were recovered from nine locations and metalaxyl-resistant isolates from three locations. A total of 104 isolates tested on the AVRDC panel resolved five physiological races. None of 42 isolates tested on the NMRIL panel caused visible infection. SSR genotyping of 127 isolates revealed 59 unique genotypes, with 42 present as singletons and 17 having 2 to 13 isolates. Isolates with identical genotypes were recovered from multiple sites across multiple years and, in many cases, had different race types or metalaxyl sensitivities. Isolates clustered into three groups with each group having almost exclusively the A1 or A2 mating type. Overall it appears long-lived genetically diverse clonal lineages are dispersed across Gansu, outcrossing is rare, and functionally important variation exists within a clonal framework.