Jing-Ze Zhang

The MAPKK FgMkk1 of Fusarium graminearum regulates vegetative differentiation, multiple stress response, and virulence via the cell wall integrity and high-osmolarity glycerol signaling pathways

Mitogen-activated protein (MAP) kinases play crucial roles in regulating fungal development, growth and pathogenicity, and in responses to the environment. In this study, we characterized a MAP kinase kinase FgMkk1 in Fusarium graminearum, the causal agent of wheat head blight. Phenotypic analyses of the FgMKK1 mutant (ΔFgMKK1) showed that FgMkk1 is involved in the regulation of hyphal growth, pigmentation, conidiation, deoxynivalenol biosynthesis and virulence of F. graminearum. ΔFgMKK1 also showed increased sensitivity to cell wall-damaging agents, and to osmotic and oxidative stresses, but exhibited decreased sensitivity to the fungicides iprodione and fludioxonil. In addition, the mutant revealed increased sensitivity to a biocontrol agent, Trichoderma atroviride. Western blot assays revealed that FgMkk1 positively regulates phosphorylation of the MAP kinases Mgv1 and FgOs-2, the key component in the cell wall integrity (CWI) and high-osmolarity glycerol (HOG) signalling pathway respectively. Yeast two-hybrid assay indicated that Mgv1 interacts with a transcription factor FgRlm1. The FgRLM1 mutant (ΔFgRLM1) showed increased sensitivity to cell wall-damaging agents and exhibited decreased virulence. Taken together, our data indicated that FgMkk1 is an upstream component of Mgv1, and regulates vegetative differentiation, multiple stress response and virulence via the CWI and HOG signalling pathways. FgRlm1 may be a downstream component of Mgv1 in the CWI pathway in F. graminearum.

In vitro Biofumigation of Brassica Tissues Against Potato Stem Rot Caused by Sclerotinia sclerotiorum

State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China (Received on November 30, 2011; Revised on May 2, 2012; Accepted on May 14, 2012)Sclerotinia sclerotiorum is a serious pathogen whichcauses yield loss in many dicotyledonous crops includ-ing potato. The objective of this study was to assess thepotential of biofumigation using three Brassica cropsincluding Brassica napus, B. juncea and B. campestrisagainst potato stem rot caused by S. sclerotiorum by invitro tests. Both macerated and irradiated dried tissueswere able to reduce radial growth and sclerotiaformation of five pathogen isolates on PDA, but mace-rated live tissues were more effective. Compared withother tested crops, B. juncea showed more inhibitoryeffect against the pathogen. The volatile compoundsproduced from macerated tissues were identified usinga gas chromatograph-mass spectrometer. The mainidentified compounds were methyl, allyl and butyl iso-thiocyanates. Different concentrations of these com-pounds inhibited mycelial growth of the pathogen invitro when applied as the vapor of pure chemicals. Anegative relationship was observed between chemicalsconcentrations and growth inhibition percentage. In thisstudy, it became clear that the tissues of local Brassicacrops release glucosinolates and have a good potential tobe used against the pathogen in field examinations. Keywords : dual culture, glucosinolates, ion trap detector,volatile compoundsPotato white mold, also known as stem rot, is a damagingdisease caused by the fungus Sclerotinia sclerotiorum. Thisdisease usually occurs in a large number of potato fieldsin Hamadan, Iran (Ojaghian, 2009) resulting in reducedyield and tuber quality. Application of fungicides, mainlyiprodione and dichloran, is the only control method beingused by local growers and it has caused environmentalconcerns leading to find non-chemical control methods suchas biofumigation.It has been shown in numerous studies that the use ofBrassica crops as green manure provides promising alter-natives to synthetic chemical fumigants (Brown and Morra,1997). Brassica crops are considered as break crops becausethey are able to break the life cycle of several soil bornepathogens such as wheat take-all (Brennan and Murray,1988). They have been proved to be effective againstnumerous nematodes (Buskov et al., 2002), fungal plantpathogens (Kirkegaard et al., 1996) and weeds (Boydston andHang, 1995). These biocontrolling effects are attributed to theproduction of volatile biotoxic compounds through a processwhich is known as biofumigation. The cruciferous tissuespossess thioglucoside compounds known as glucosinolates(GSLs), the secondary polar sulfur metabolites, which are thereason for the sharp or biting taste of condiments or theflavors of different edible Brassica tissues (McGregor et al.,1983). The GSLs are not harmful to organisms by themselvesbut they can change into isothiocyanates (ITCs), nitriles,thiocyanates, and oxazolidinethiones and other toxic com-pounds (Bending and Lincoln, 1999) by the endogenousenzyme myrosinase when Brassica tissues are damaged(Bones and Rossiter, 1996). The quality of the hydrolysisproducts depends on the type of organic side chain on theparent molecule and the environmental conditions (Rosa etal., 1997). No research has been conducted on the presence andidentification of GSLs in Iranian cultivars of Brassicas. Theobjective of this study was to evaluate the potential of bio-fumigation using three local Brassica crops against a numberof S. sclerotiorum isolates by in vitro experiments.Firstly, the inhibitory effects of Brassica tissues on radialgrowth and sclerotia formation of S. sclerotiorum weredetermined. Five highly aggressive isolates of S. sclerotiorum(Sc18d; Bahar, Sd6b; Bahar, Sd21n; Lalehjin, Sc59k; Baharand Sa21b; Lalehjin) were obtained from the CultureCollection of Plant Pathology Laboratory, Agriculture College,BuAli Sina University, Hamadan, Iran. These isolates hadalready been sampled in five heavily infected potato fields inHamadan province. The isolates were routinely cultured onpotato dextrose agar (PDA, 39 g/L; Merck, Darmstadt,Germany) and stored at 4 ± 1

Biology of Colletotrichum horii, the causal agent of persimmon anthracnose

Colletotrichum horii causes serious anthracnose on persimmon (Diospyros kaki cv. Wuheshi). The taxon was previously identified as C. gloeosporioides and only recently revealed to be C. horii based on molecular data and comparisons to type specimens. This fungus provides an important new model for examining plant–fungus interactions in the perennial persimmon crop. In this paper, we review available information on C. horii, with special focus on symptoms, morphological characteristics, phylogenetic analysis, host-specificity and pathogenicity testing, infection processes, and the effects of environment factors on anthracnose development, including a discussion on future prospects.

Inhibitory effect and enzymatic analysis of E-cinnamaldehyde against sclerotinia carrot rot.

This study was conducted to determine the inhibitory effect of E-cinnamaldehyde (EC) against causal agent of storage carrot rot, Sclerotinia sclerotiorum, under in vivo and in vitro conditions. Based on the results, EC was able to completely inhibit mycelial growth of three isolates (P>0.05) in both volatile and contact phases after 6days at the concentrations 200μl and 1μl/ml, respectively. In addition, EC at concentrations 1 and 10μl/ml completely inhibited carpogenic germination of three isolates. The results of in vivo trials showed that EC at the concentration of 10μl/ml was able to control the disease caused by isolates 1 and 3. However the disease caused by isolate 2 was inhibited with the concentration of 20μl/ml. In enzyme analyses, the activity of polyphenoloxidase and peroxidase did not change in the inoculated carrots after application of EC. Furthermore, the level of phenylalanine ammonia lyase decreased. These results indicated that EC does not have any potential to be considered as resistance inducers against sclerotinia carrot rot.

Early detection of white mold caused by Sclerotinia sclerotiorum in potato fields using real-time PCR

Potato white mold caused by Sclerotinia sclerotiorum is an important plant disease occurring in many potato-producing areas worldwide. This disease causes considerable yield losses in a large number of potato fields of Hamedan province, Iran. Although several non-chemical methods have been suggested against the pathogen, application of chemicals is considered a major method employed by the growers in Hamedan fields leading to considerable pollution of underground water. Therefore, it is necessary to find techniques to help growers make a more timely decision for chemical application. In this research, a pair of PCR primers was designed according to the nucleotide sequence of a region of DNA amplified by the primer M13. This primer set amplified a 352-bp fragment from all isolates of S. sclerotiorum collected from Hamedan, Iran, and Hangzhou, China. However, this situation was not observed in other tested fungi. A real-time PCR assay was conducted to detect early infection of the pathogen on potato petals. This real-time PCR assay is suggested for growers to make timely decisions on chemical application.

Ultrastructure and phylogeny of Ustilago coicis

Ustilago coicis causes serious smut on Coix lacryma-jobi in Dayang Town, Jinyun County, Zhejiang Province of China. In this paper, ultrastructural assessments on fungus-host interactions and teliospore development are presented, and molecular phylogenetic analyses have been done to elucidate the phylogenetic placement of the taxon. Hyphal growth within infected tissues was both intracellular and intercellular and on the surface of fungus-host interaction, and the fungal cell wall and the invaginated host plasma membrane were separated by a sheath comprising two distinct layers between the fungal cell wall and the invaginated host plasma membrane. Ornamentation development of teliospore walls was unique as they appeared to be originated from the exosporium. In addition, internal transcribed spacer (ITS) and large subunit (LSU) sequence data showed that U. coicis is closely related to Ustilago trichophora which infects grass species of the genus Echinochloa (Poaceae).

Antagonistic interaction between Trichoderma asperellum and Phytophthora capsici in vitro

Phytophthora capsici is a phytopathogen that causes a destructive pepper blight that is extremely difficult to control. Using a fungicide application against the disease is costly and relatively ineffective and there is also a huge environmental concern about the use of such chemicals. The genus Trichoderma has been known to have a potential biocontrol issue. In this paper we investigate the mechanism for causing the infection of T. asperellum against P. capsici. Trichoderma sp. (isolate CGMCC 6422) was developed to have a strong antagonistic action against hyphae of P. capsici through screening tests. The strain was identified as T. asperellum through using a combination of morphological characteristics and molecular data. T. asperellum was able to collapse the mycelium of the colonies of the pathogen through dual culture tests by breaking down the pathogenic hyphae into fragments. The scanning electron microscope showed that the hyphae of T. asperellum surrounded and penetrated the pathogens hyphae, resulting in hyphal collapse. The results show that seven days after inoculation, the hyphae of the pathogen were completely degraded in a dual culture. T. asperellum was also able to enter the P. capsici oospores through using oogonia and then developed hyphae and produced conidia, leading to the disintegration of the oogonia and oospores. Seven days after inoculation, an average 10.8% of the oospores were infected, but at this stage, the structures of oospores were still intact. Subsequently, the number of infected oospores increased and the oospores started to collapse. Forty-two days after inoculation, almost all the oospores were infected, with 9.3% of the structures of the oospores being intact and 90.7% of the oospores having collapsed.概要目 的筛选出对辣椒疫霉菌具有高效拮抗作用的木霉生防菌株, 研究其对辣椒菌菌丝体和卵孢子的作用机制, 评价其应用于辣椒疫病的生防潜力。创新点首次在超微结构水平上报道棘孢木霉菌菌丝能重寄生于辣椒疫霉菌的卵孢子, 为木霉生防菌的应用提供了科学理论依据。方 法从土壤中分离木霉菌株, 采用对峙培养法筛选木霉生防菌株。通过形态学和多基因序列 (ITS、 tef1 和 rpb2) 进行鉴定, 明确获得木霉菌株的种类。 通过细胞学和超微结构观察, 研究木霉生防菌对辣椒疫霉菌菌丝和卵孢子的拮抗机制。结 论本研究筛选出了对辣椒疫霉菌菌丝具有高效拮抗作用的一个木霉菌株 (CGMCC 6422), 被鉴定为棘孢木霉菌 (Trichoderma asperellum)。 细胞学和超微结构显示, 该菌株能塌陷辣椒疫霉菌的菌落, 通过缠绕和穿透辣椒疫霉菌的菌丝体, 引起菌丝体解体; 首次观察到该菌株能侵染辣椒疫霉菌的卵孢子, 并引起卵孢子完全降解。 综上所述, 筛选出的木霉生防菌株 CGMCC 6422 具有应用于防治辣椒疫病的生防潜力。

A new species of Scopulariopsis and its synergistic effect on pathogenicity of Verticillium dahliae on cotton plants

A new species, Scopulariopsis gossypii, was found to be present in the vascular bundles of cotton plants (Gossypium hirsutum) infected by Verticillium dahliae which is an economically important pathogen in Hangzhou, China. The fungus was only present in the diseased plants, but it never became isolated from the healthy plants. Scanning electron microscopy showed that the fungus was present in parenchyma cells enclosing vessels in dark brown vascular tissues of stems, and produced asexual conidia within the tissues. Phylogenetic analysis of combined nuclear ribosomal D1/D2 region of the 28S rDNA as well as translation elongation factor 1-alpha (TEF1) and beta-tubulin (TUB) gene showed that S. gossypii represents an undescribed species of Scopulariopsis, Microascaceae. In this study, characteristics of sexual and asexual stages of the fungus were described, illustrated and compared with similar taxa. In addition, the molecular and morphological analyses indicated that S. gossypii was a distinct species of Scopulariopsis. The pathogenicity tests proved by inoculation of wounded roots confirmed that S. gossypii was an opportunistic pathogen causing leaf interveinal chlorosis and vascular browning of cotton plants. However S. gossypii did not infect host with undamaged roots. Moreover, coinoculation with S. gossypii and V. dahliae significantly increased disease severity.