Isolation and Identification of Pathogens Causing Marigold (Tagetes erecta L.) Black Spot in Beijing,China

Black spot leads to great marigold losses worldwide. The disease is characterized by black spots on leaves and stems in its early stages, and the whole plant has black rot at the advanced stage. In this report, 6 of 217 Alternaria strains isolated from lesions of marigold plants in Beijing were randomly selected. The morphological characteristics and a pathogenic tree based on two protein‐coding genes (gpd and alt a 1) indicated that Alternaria tagetica is the causal agent of marigold black spot in Beijing. All six Alternaria strains could successfully re‐infect marigold, but they could not infect carrot or zinnia by either spore spray in a greenhouse or planting experiments in the epidemic area. This is the first report of the A. tagetica pathogen being isolated from marigold in Beijing.     

Identification and Pathogenicity of Rhizoctonia spp. Causing Wirestem of Betula nigra in China

During July 2004, wirestem was frequently observed on the seedlings of Betula nigra at Dehong district in Yunnan Province, China. Isolates of Rhizoctonia spp. consistently obtained from their diseased leaves, roots and stems were identified as belonging to binucleate Rhizoctonia anastomosis groups (AG) AG‐P and AG‐R, and R. solani AG‐I IB and AG‐4 HG‐I, based on cultural characteristics, nuclear staining, anastomisis reaction and analysis of their ITS rDNA region. The percentage of recovery of AG‐P, AG‐1, AG‐R and AG‐4 was 48%, 39%, 8% and 3%, respectively. This is the first report of wirestem of red birch cause by binucleate Rhizoctonia AG‐P and AG‐R, and R. solani AG‐1 IB and AG‐4 HG‐I in China.     

Characterization and Pathogenicity of Lasiodiplodia theobromae Causing Black Root Rot and Identification of Novel Sources of Resistance in Mulberry Collections

Black root rot (BRR) caused by Lasiodiplodia theobromae is an alarming disease of mulberry that causes tremendous economic losses to sericulture farmers in India and China. Successful control of this disease can be attained by screening germplasm and identifying resistant sources. Seventy four diseased root samples were collected from farmer’s fields belonging to four major mulberry growing states of South India. Based on morpho-cultural and scanning electron microscopy studies, 57 fungal isolates were characterized and identified as L. theobromae. Phylogenetic analysis of concatenated internal transcribed spacer and β-tubulin sequences revealed variation of the representative 20 isolates of L. theobromae. Following the root dip method of inoculation, pathogenicity studies on susceptible mulberry genotypes (Victory-1 and Thailand male) recognized the virulent isolate MRR-142. Accordingly, MRR-142 isolate was used to evaluate resistance on a set of 45 diverse mulberry accessions. In the repeated experiments, the mulberry accession ME-0168 which is an Indonesian origin belonging to Morus latifolia was found to be highly resistant consistently against BRR. Eight accessions (G2, ME-0006, ME-0011, ME-0093, MI-0006, MI-0291, MI-0489, and MI-0501) were found to be resistant. These promising resistant resources may be exploited in mulberry breeding for developing BRR resistant varieties and to develop mapping populations which successively helps in the identification of molecular markers associated with BRR.     

Microbial Pathogens in the Fungal Kingdom

The fungal kingdom is vast, spanning ~1.5 to as many as 5 million species diverse as unicellular yeasts, filamentous fungi, mushrooms, lichens, and both plant and animal pathogens. The fungi are closely aligned with animals in one of the six to eight supergroups of eukaryotes, the opisthokonts. The animal and fungal kingdoms last shared a common ancestor ~1 billion years ago, more recently than other groups of eukaryotes. As a consequence of their close evolutionary history and shared cellular machinery with metazoans, fungi are exceptional models for mammalian biology, but prove more difficult to treat in infected animals. The last common ancestor to the fungal/metazoan lineages is thought to have been unicellular, aquatic, and motile with a posterior flagellum, and certain extant species closely resemble this hypothesized ancestor. Species within the fungal kingdom were traditionally assigned to four phyla, including the basal fungi (Chytridiomycota, Zygomycota) and the more recently derived monophyletic lineage, the dikarya (Ascomycota, Basidiomycota). The fungal tree of life project has revealed that the basal lineages are polyphyletic, and thus there are as many as eight to ten fungal phyla. Fungi that infect vertebrates are found in all of the major lineages, and virulence arose multiple times independently. A sobering recent development involves the species Batrachochytrium dendrobatidis from the basal fungal phylum, the Chytridiomycota, which has emerged to cause global amphibian declines and extinctions. Genomics is revolutionizing our view of the fungal kingdom, and genome sequences for zygomycete pathogens (Rhizopus, Mucor), skin-associated fungi (dermatophytes, Malassezia), and the Candida pathogenic species clade promise to provide insights into the origins of virulence. Here we survey the diversity of fungal pathogens and illustrate key principles revealed by genomics involving sexual reproduction and sex determination, loss of conserved pathways in derived fungal lineages that are retained in basal fungi, and shared and divergent virulence strategies of successful human pathogens, including dimorphic and trimorphic transitions in form. The overarching conclusion is that fungal pathogens of animals have arisen repeatedly and independently throughout the fungal tree of life, and while they share general properties, there are also unique features to the virulence strategies of each successful microbial pathogen.     

Identification and Characterization of Fungal Pathogens Associated with Boxwood Diseases in the Republic of Korea

Boxwood is a representative ornamental shrub that is widely used in landscaping horticulture. After pruning, damaged leaves or stems of boxwoods are unavoidably vulnerable to infection by various plant pathogens. Several boxwood diseases caused by fungi, such as Volutella blight and Macrophoma leaf spot, have been reported worldwide including Republic of Korea. In this study, we isolated and identified fungal pathogens of boxwood diseases that occurred in Korea and characterized their morphological and taxonomic characteristics. Boxwood samples showing blight symptoms were collected in Seoul, Republic of Korea, and the putative fungal pathogens Pseudonectria buxi, P. foliicola, and Neofusicoccum buxi were successfully identified. Investigation of the morphological features of the field isolates, including mycelial growth and conidial morphology, and phylogenetic analysis of multiple DNA barcode loci revealed that there were some morphological and genetic variations among isolates, but all of the analyzed isolates were closely related to the corresponding reference strains. We also found that P. foliicola strains were more virulent than P. buxi, and the N. buxi strains isolated in this study were weak pathogens or saprophytes. The results of our study will contribute to the development of control strategies for boxwood diseases caused by fungi and accelerate research on the complex ecology of boxwood diseases.     

多重PCR对真菌性角膜炎主要致病菌的菌属鉴定

目的:建立多重PCR体系对真菌性角膜炎主要致病真菌进行快速诊断并同时进行菌属鉴定的方法。方法:建立两个多重PCR体系(体系1和体系2),对真菌性角膜炎九种主要致病真菌DNA进行检测,观察该体系对真菌临床菌株、人类基因组及其他眼部常见致病微生物DNA的检测结果。结果:体系1对镰孢菌属扩增均产生约360bp的特异产物,对曲霉菌属、牵连青霉菌和新月弯孢菌扩增均产生约470bp的特异产物。体系2对镰孢菌属、曲霉菌属均无特异产物,而对牵连青霉菌产生了360bp的特异产物,对新月弯孢霉产生了300bp的特异产物。根据DNA模板在两个多重PCR体系中扩增出的不同特异条带可将九种真菌分为四个菌属。57株真菌临床菌株中55株的鉴定结果与常规鉴定结果一致。两体系对人类基因组及其他眼部常见致病微生物DNA的扩增结果均为阴性。结论:通过两个多重PCR体系检测可将真菌性角膜炎在菌属水平进行诊断及鉴定。该方法具有快速、简便、特异、灵敏的特点,具有较好的临床应用前景。     

Responses of Wheat Yield,Macro- and Micro-Nutrients,and Heavy Metals in Soil and Wheat following the Application of Manure Compost on the North China Plain

The recycling of livestock manure in cropping systems is considered to enhance soil fertility and crop productivity. However, there have been no systematic long-term studies of the effects of manure application on soil and crop macro- and micro-nutrients, heavy metals, and crop yields in China, despite their great importance for sustainable crop production and food safety. Thus, we conducted field experiments in a typical cereal crop production area of the North China Plain to investigate the effects of compost manure application rates on wheat yield, as well as on the macro-/micro-nutrients and heavy metals contents of soil and wheat. We found that compost application increased the soil total N and the available K, Fe, Zn, and Mn concentrations, whereas the available P in soil was not affected, and the available Cu decreased. In general, compost application had no significant effects on the grain yield, biomass, and harvest index of winter wheat. However, during 2012 and 2013, the N concentration decreased by 9% and 18% in straw, and by 16% and 12% in grain, respectively. With compost application, the straw P concentration only increased in 2012 but the grain P generally increased, while the straw K concentration tended to decrease and the grain K concentration increased in 2013. Compost application generally increased the Fe and Zn concentrations in straw and grain, whereas the Cu and Mn concentrations decreased significantly compared with the control. The heavy metal concentrations increased at some compost application rates, but they were still within the safe range. The balances of the macro-and micro-nutrients indicated that the removal of nutrients by wheat was compensated for by the addition of compost, whereas the level of N decreased without the application of compost. The daily intake levels of micronutrients via the consumption of wheat grain were still lower than the recommended levels when sheep manure compost was applied, except for that of Mn.     

Expression of Lignin Biosynthetic Genes in Wheat during Development and upon Infection by Fungal Pathogens

As a major component of the cell wall, lignin plays an important role in plant development and defense response to pathogens, but negatively impacts biomass processability for biofuels. Silencing the target lignin genes for greater biomass processability should not significantly affect plant development and biomass yield but also must not compromise disease resistance. Here, we report experiments to identify a set of lignin genes that may be silenced without compromising disease resistance. We profiled the expression of 32 lignin biosynthetic candidate genes by qRT-PCR in 17 wheat tissues collected at three developmental stages. Twenty-one genes were expressed at a much higher level in stems compared to sheaths and leaf blades. Expression of seven these genes significantly correlated with lignin content. The co-expression patterns indicated that these 21 genes are under strong developmental regulation and may play a role in lignin biosynthesis. Profiling gene expression of same tissues challenged by two fungal pathogens, Fusarium graminearum and Puccina triticina indicated that expression of 17 genes was induced by F. graminearum. Only PAL1, a non-developmental-regulated gene, was induced by P. triticina. Thus, lignin biosynthetic pathway overlaps defense response to F. graminearum. Based on these criteria, 17 genes, F5H1, F5H2, 4CL2, CCR2, COMT1, and COMT2 in particular that do not overlap with disease resistance pathway, may be the targets for downregulation.     

Effects of Fungal Root Pathogens on the Population Dynamics of Biocontrol Strains of Fluorescent Pseudomonads in the Wheat Rhizosphere

The influences of Gaeumannomyces graminis var. tritici (which causes take-all of wheat), Rhizoctonia solani AG-8 (which causes rhizoctonia root rot of wheat), Pythium irregulare, P. aristosporum, and P. ultimum var. sporangiiferum (which cause pythium root rot of wheat) on the population dynamics of Pseudomonas fluorescens 2-79 and Q72a-80 (bicontrol strains active against take-all and pythium root rot of wheat, respectively) in the wheat rhizosphere were examined. Root infection by either G. graminis var. tritici or R. solani resulted in populations of both bacterial strains that were equal to or significantly larger than their respective populations maintained on roots in the absence of these pathogens. In contrast, the population of strain 2-79 was significantly smaller on roots in the presence of any of the three Pythium species than on noninfected roots and was often below the limits of detection (50 CFU/cm of root) on Pythium-infected roots after 40 days of plant growth. In the presence of either P. aristosporum or P. ultimum var. sporangiiferum, the decline in the population of Q72a-80 was similar to that observed on noninfected roots; however, the population of this strain declined more rapidly on roots infected by P. irregulare than on noninfected roots. Application of metalaxyl (which is selectively inhibitory to Pythium spp.) to soil naturally infestated with Pythium spp. resulted in significantly larger rhizosphere populations of the introduced bacteria over time than on plants grown in the same soil without metalaxyl. It is apparent that root infections by fungal pathogens may either enhance or depress the population of fluorescent pseudomonads introduced for their control, with different strains of pseudomonads reacting differentially to different genera and species of the root pathogens.     

河北省冬枣黑斑病病原菌的分离鉴定及生物防治初探

冬枣黑斑病是冬枣重要病害之一,目前多以化学农药进行防治,给自然环境和人类健康带来了严重的威胁。河北省是冬枣种植大省,进行冬枣黑斑病病原菌的分离鉴定对冬枣黑斑病的有效防治具有重要意义。2014年8月至10月从河北省沧州、衡水、邯郸、邢台等地采集冬枣黑斑病病果,采用PDA培养基进行病原菌分离,从病样中共分离出2株分离频率较高的真菌,经过回接和再分离实验筛选出河北省冬枣黑斑病的致病菌株,经形态学和ITS序列分析初步确定该菌为细交链格孢(Alternaria alternata)。以枯草芽胞杆菌J18进行冬枣采后黑斑病的防治,浓度为1×108cfu/m L的菌液对病害的防效为80.67%。     

Identification of some Bacteria from Paddy Antagonistic to Several Rice Fungal Pathogens

The effect of 23 bacterial strains from ricefields in the tropics on rice seed germination and on radicle and hypocotyl development of four rice cultivars was determined. There was a varietal difference in response to seed bacterization with the different bacterial strains. Germination of cv. IR58 increased from 78 to 93 %, that of cv. IR64, from 89 to 97 %. Less effects on germination of cvs IR42 and IR36 were observed. All strains inhibited the mycelial growth of Rhizoctonia solani in vitro. The three strains, identified as Bacillus subtilis, inhibited the mycelial growth of eight fungal pathogens whereas the other strains were pathogen-specific. Seed bacterization with these bacterial strains provided a sheath blight protection of 4. 5 to 73 % in the glasshouse trial. These 23 bacterial strains were identified by phenotypic tests using the API systems, morphological and biochemical features, and by comparison of electrophoretic patterns after sodium dodecyl sulphate polyacrylamide gel electrophoresis. Bacterial strains were identified (number of strains in brackets) as: Bacillus subtilis (3), Bacillus laterosporus (1), Bacillus pumilus (1), Pseudomonas aeruginosa (7), Pseudomonas belonging to section 1 (5), Erwina herbicola-like (1), and Serratia marcescens (1). The features of the other four strains were similar to Serratia except for the DNAase and lipase activities.     

群体感应与病原菌致病性研究进展

群体感应是细菌根据细胞密度变化进行基因表达调控的一种生理行为。当细菌密度达到临界阈值时能释放一些特定的自诱导信号分子,从而调节本种群或同环境中其他种群的群体行为。细菌群体感应参与包括人类、动植物、病原菌在内的多种生物的生物学功能调节,如生物膜的形成、毒力因子的产生、病原菌的耐药性等。深入研究病原菌群体感应系统的调控机制,将提高对病原菌发病机制的认识,有利于以群体感应作为防治疾病策略的研究。系统阐述了群体感应系统的组成类型、群体感应与病原菌致病性的关系,及其在抑制病原菌致病方面的应用。     

Identification of Fungal Pathogens by Visible Microarray System in Combination with Isothermal Gene Amplification

The increasing incidence of infectious diseases caused by fungi in immunocompromised patients has encouraged researchers to develop rapid and accurate diagnosis methods. Identification of the causative fungal species is critical in deciding the appropriate treatment, but it is not easy to get satisfactory results due to the difficulty of fungal cultivation and morphological identification from clinical samples. In this study, we established a microarray system that can identify 42 species from 24 genera of clinically important fungal pathogens by using a chemical color reaction in the detection process. The array uses the internal transcribed spacer region of the rRNA gene for identification of fungal DNA at the species level. The specificity of this array was tested against a total of 355 target and nontarget fungal species. The fungal detection was succeeded directly from 10³ CFU/ml for whole blood samples, and 50 fg DNA per 1 ml of serum samples indicating that the array system we established is sensitive to identify infecting fungi from clinical sample. Furthermore, we conducted isothermal amplification in place of PCR amplification and labeling. The successful identification with PCR-amplified as well as isothermally amplified target genes demonstrated that our microarray system is an efficient and robust method for identifying a variety of fungal species in a sample.     

Response of CH4 and N2O Emissions and Wheat Yields to Tillage Method Changes in the North China Plain

The objective of this study was to quantify soil methane (CH₄) and nitrous oxide (N₂O) emissions when converting from minimum and no-tillage systems to subsoiling (tilled soil to a depth of 40 cm to 45 cm) in the North China Plain. The relationships between CH₄ and N₂O flux and soil temperature, moisture, NH₄ ⁺-N, organic carbon (SOC) and pH were investigated over 18 months using a split-plot design. The soil absorption of CH₄ appeared to increase after conversion from no-tillage (NT) to subsoiling (NTS), from harrow tillage (HT) to subsoiling (HTS) and from rotary tillage (RT) to subsoiling (RTS). N₂O emissions also increased after conversion. Furthermore, after conversion to subsoiling, the combined global warming potential (GWP) of CH₄ and N₂O increased by approximately 0.05 kg CO₂ ha⁻¹ for HTS, 0.02 kg CO₂ ha⁻¹ for RTS and 0.23 kg CO₂ ha⁻¹ for NTS. Soil temperature, moisture, SOC, NH₄ ⁺-N and pH also changed after conversion to subsoiling. These changes were correlated with CH₄ uptake and N₂O emissions. However, there was no significant correlation between N₂O emissions and soil temperature in this study. The grain yields of wheat improved after conversion to subsoiling. Under HTS, RTS and NTS, the average grain yield was elevated by approximately 42.5%, 27.8% and 60.3% respectively. Our findings indicate that RTS and HTS would be ideal rotation tillage systems to balance GWP decreases and grain yield improvements in the North China Plain region.     

Grain Yield and Water Use Efficiency in Extremely-Late Sown Winter Wheat Cultivars under Two Irrigation Regimes in the North China Plain

Wheat production is threatened by water shortages and groundwater over-draft in the North China Plain (NCP). In recent years, winter wheat has been increasingly sown extremely late in early to mid-November after harvesting cotton or pepper. To improve water use efficiency (WUE) and guide the extremely late sowing practices, a 3-year field experiment was conducted under two irrigation regimes (W1, one-irrigation, 75 mm at jointing; W2, two-irrigation, 75 mm at jointing and 75 mm at anthesis) in 3 cultivars differing in spike size (HS4399, small spike; JM22, medium spike; WM8, large spike). Wheat was sown in early to mid-November at a high seeding rate of 800–850 seeds m⁻². Average yields of 7.42 t ha⁻¹ and WUE of 1.84 kg m⁻³ were achieved with an average seasonal evapotranspiration (ET) of 404 mm. Compared with W2, wheat under W1 did not have yield penalty in 2 of 3 years, and had 7.9% lower seasonal ET and 7.5% higher WUE. The higher WUE and stable yield under W1 was associated with higher 1000-grain weight (TGW) and harvest index (HI). Among the 3 cultivars, JM22 had 5.9%–8.9% higher yield and 4.2%–9.3% higher WUE than WM8 and HS4399. The higher yield in JM22 was attributed mainly to higher HI and TGW due to increased post-anthesis biomass and deeper seasonal soil water extraction. In conclusion, one-irrigation with a medium-sized spike cultivar JM22 could be a useful strategy to maintain yield and high WUE in extremely late-sown winter wheat at a high seeding rate in the NCP.     

福建厦门地区蚜虫病原真菌调查及其发生规律

从2014年至2015年对厦门地区蚜虫虫霉发生和流行情况进行初步调查,共鉴定出6种虫霉目病原真菌:新蚜虫疠霉Pandora neoaphidis、努利虫疠霉Pandora nouryi、普朗肯虫霉Entomophthora planchoniana、暗孢耳霉Conidiobolus obscurus、弗雷生新接霉Neozygites fresenii、根虫瘟霉Zoophthora radicans。新蚜虫疠霉和普朗肯虫霉是春季蔬菜蚜虫种群中发生、流行的最重要病原真菌,夏季未观察到虫霉的发生。     

华北平原冬小麦农田蒸散量

以华北平原冬小麦农田为研究对象,采用涡度相关技术和热红外遥感技术,研究了不同环境条件下土壤含水量与农田蒸散量及作物冠层温度的关系.结果表明,冬小麦在农田郁闭(LAI≥3)、晴天和土壤相对含水量低于田间持水量65%的情况下,蒸发比值日变化正午前后出现相对较低且平稳的变化趋势.在晴天情况下,农田潜热通量与作物冠层温度日变化和季节变化均呈极显著的非线性相关关系,而冠气温差、农田相对蒸散量则与0～100 cm土层的土壤相对含水量密切相关.以13:30～14:00的平均冠层温度值T_c、日最高气温T_{a max}和日净辐射总量Rn_d为统计数据,确立了冬小麦农田日蒸散量ET_d （mm）估算简化模式参数.     

The Black Yeast Exophiala dermatitidis and Other Selected Opportunistic Human Fungal Pathogens Spread from Dishwashers to Kitchens

We investigated the diversity and distribution of fungi in nine different sites inside 30 residential dishwashers. In total, 503 fungal strains were isolated, which belong to 10 genera and 84 species. Irrespective of the sampled site, 83% of the dishwashers were positive for fungi. The most frequent opportunistic pathogenic species were Exophiala dermatitidis, Candida parapsilosis sensu stricto, Exophiala phaeomuriformis, Fusarium dimerum, and the Saprochaete/Magnusiomyces clade. The black yeast E. dermatitidis was detected in 47% of the dishwashers, primarily at the dishwasher rubber seals, at up to 10⁶ CFU/cm²; the other fungi detected were in the range of 10² to 10⁵ CFU/cm². The other most heavily contaminated dishwasher sites were side nozzles, doors and drains. Only F. dimerum was isolated from washed dishes, while dishwasher waste water contained E. dermatitidis, Exophiala oligosperma and Sarocladium killiense. Plumbing systems supplying water to household appliances represent the most probable route for contamination of dishwashers, as the fungi that represented the core dishwasher mycobiota were also detected in the tap water. Hot aerosols from dishwashers contained the human opportunistic yeast C. parapsilosis, Rhodotorula mucilaginosa and E. dermatitidis (as well as common air-borne genera such as Aspergillus, Penicillium, Trichoderma and Cladosporium). Comparison of fungal contamination of kitchens without and with dishwashers revealed that virtually all were contaminated with fungi. In both cases, the most contaminated sites were the kitchen drain and the dish drying rack. The most important difference was higher prevalence of black yeasts (E. dermatitidis in particular) in kitchens with dishwashers. In kitchens without dishwashers, C. parapsilosis strongly prevailed with negligible occurrence of E. dermatitidis. F. dimerum was isolated only from kitchens with dishwashers, while Saprochaete/Magnusiomyces isolates were only found within dishwashers. We conclude that dishwashers represent a reservoir of enriched opportunistic pathogenic species that can spread from the dishwasher into the indoor biome.