Reduction of clubroot (Plasmodiophora brassicae) formation in Arabidopsis thaliana after treatment with prohexadione‐calcium,an inhibitor of oxoglutaric acid‐dependent dioxygenases

Recently, flavonoids were shown to modulate the outcome of clubroot development in Arabidopsis thaliana after infection with the obligate biotrophic pathogen Plasmodiophora brassicae. Therefore, the development of clubroot disease was investigated in Arabidopsis after treatment with prohexadione‐calcium (ProCa), an inhibitor of ascorbic acid/2‐oxoglutaric acid‐dependent dioxygenases such as flavanone‐3‐hydroxylase. The treatment resulted in a reduction of the flavonols quercetin and kaempferol in clubroots, whereas the precursor naringenin highly accumulated. The root system of ProCa‐treated plants was better developed although galls were still visible. Thus, ProCa treatment resulted in reduced gall size. Flavonoids are thought to inhibit polar auxin transport by modulating auxin efflux carriers. It was investigated whether the auxin response might change as a consequence of the accumulation of naringenin in ProCa‐treated plants. In the areas of gall development an auxin response was indicated by the auxin‐responsive promoter DR5 coupled to the reporter β‐glucuronidase (GUS), whereas very little staining was found in healthy root parts. No differences in GUS activity were found between P. brassicae‐infected and ProCa‐treated plants, and plants only infected with P. brassicae, indicating that the effect of ProCa treatment on clubroot reduction is not via changes in auxin responses. As ProCa is also an inhibitor of late steps in gibberellin biosynthesis, a specific gibberellin biosynthesis inhibitor, chlormequatchloride (CCC), was tested on club development. However, CCC did not reduce disease symptoms, indicating that the observed reduced gall development was not because of gibberellin biosynthesis inhibition by ProCa.     

Susceptibility of hairy root lines of Brassica species to Plasmodiophora brassicae and in an in vitro subculture system

To investigate the susceptibility of hairy root lines of Brassica species to Plasmodiophora brassicae, hairy roots were induced in a number of Brassica species with Agrobacterium rhizogenes. Turnip hairy root was highly susceptible to P. brassicae; infection rates were high and large galls formed. In contrast, the rates of root hair infection and gall formation on intact Brassica plants did not differ significantly from the control. To induce resting spore formation, turnip hairy roots were incubated at 15°, 20°, or 25°C after 3 weeks of incubation at 25°C. The number and fresh mass of the galls per hairy root were higher and formation of resting spores was greatest after a 7-week incubation at 20°C. To subculture P. brassicae using turnip hairy root, turnip hairy roots were reinoculated with resting spores and gall with resting spores then formed on the hairy roots. In this way, P. brassicae using hairy roots could be subcultured in vitro two or three times on three single-spore isolates of P. brassicae. This is the first report of in vitro subculture of P. brassicae using hairy root.     

Histological analysis of spindle and spheroid root galls caused by Plasmodiophora brassicae

Clubroot of crucifers, caused by the obligate parasite Plasmodiophora brassicae, is characterized by the formation of conspicuous root galls. These galls usually have a club- or spindle-shaped morphology, and interfere with water and nutrient uptake by infected plants. Smaller galls, historically regarded as resistance structures and distinct from the typical spindle-shaped galls, have also been identified and termed ‘spheroid galls’ because of their spherical or nearly spherical form. An assessment of various Brassica species and varieties revealed that spheroid galling could be observed in all genotypes investigated, but occurred regularly only in a few particular host/P. brassicae combinations. While spindle gall formation was coincident with the expansion of the stele and infection of secondary tissues by P. brassicae, spheroid galls typically had a region of proliferating tissue that corresponded to the secondary cortex and periderm of the healthy plants, with the outer proliferating tissue less infected than the inner portions. The underlying host tissue showed limited secondary tissue development, was largely uninfected, and, where infection occurred, a continuous stele was maintained. An active host defensive reaction, in the form of cell lignification or the hypersensitive response, was not observed, while pathogen resting spores were visible in one longitudinal section of a spheroid gall. These findings suggest that while the proliferation of P. brassicae is restricted in spheroid galls, these structures are not indicative of complete resistance to clubroot.     

Analysis of the expression patterns of the Arabidopsis thaliana tubulin-1 and Zea mays ubiquitin-1 promoters in rice plants in association with nematode infection

The temporal and spatial expression of three promoters was investigated in transgenic rice plants using promoter-β-glucuronidase (gusA) reporter gene fusions. The promoters studied were ubiquitin-1 (UBI-1) of Zea mays, Cauliflower Mosaic Virus 35S gene (CaMV35S) and a tubulin gene (TUB-1) of Arabidopsis thaliana. The TUB-1 promoter provided 7.32-fold more GUS activity in roots relative to tillers. This was significantly different from the corresponding value of 2.82-fold for CaMV35S but not from that of 4.55-fold for UBI-1, activity of both promoters was higher in the root tips and zone of elongation than mature roots. This younger root tissue represented a declining proportion of the expanding root system with time. Older tissue expressing GUS under control of the TUB-1 promoter showed a steeper decline in activity with time than occurred with the UBI-1 promoter. Nematode infection did not alter the overall pattern of expression from the two promoters, except that the giant cells induced by Meloidogyne incognita retained TUB-1 promoter activity as roots matured. Pratylenchus zeae invaded older root regions than M. incognita and no changes in promoter activity were detected where it fed. The results suggest the TUB-1 promoter has characteristics that favour its use for delivering anti-feedants, such as cysteine proteinase inhibitors, to M. incognita.     

Histological methods to detect the clubroot pathogen Plasmodiophora brassicae during its complex life cycle

The clubroot pathogen Plasmodiophora brassicae is an obligate biotrophic protist that lives in close relationship with its host cell. The roots of the host plants are colonized and the plant growth is altered upon infection. While shoots can be stunted and show wilt symptoms after longer infection periods, the root system is converted to a tumorous root tissue, called ‘clubroot’, by alterations of the plant growth promoting hormones auxin, cytokinin and brassinosteroid. Because the life cycle occurs largely within the host cells, this leads to dramatic changes in host root morphology and anatomy. Thus, the identification of the respective protist structures in the host tissue by microscopy is challenging. Different staining methods as well as fluorescence and electron microscopy of thin sections can reveal specific life stages of P. brassicae and can yield additional information on the changes in the host tissues concerning, for example, cell wall properties. In addition, promoter–reporter fusions, immunostaining methods and in situ hybridization techniques can be used to gain additional information on the changes in the host roots.     

基于根肿菌早期侵染量的白菜抗性分析

为探究不同抗性白菜品种对根肿菌侵染量的影响,通过水培法观测4个白菜品种接种根肿菌后其在寄主根内的发展动态,实时荧光定量PCR检测技术对同期根内含菌量进行分析,并结合温室盆栽试验获得的各品种发病数据进行相关性分析。结果表明,早熟长江5号根肿病的发病率和病情指数分别为82.5%和36.2,华良早五号为66.7%和37.5,CR-春美为68.9%和36.0,可归为感病品种,而CR-英雄为52.5%和16.5,可归为抗病品种;水培法接种观测和PCR定量结果显示,根毛侵染率、皮层侵染数以及根内含菌量均表现为CR-英雄最低,显著低于其它3个品种。根毛侵染率、皮层侵染数、根内含菌量、发病率四者间呈显著相关,其R2最大值变幅为0.84~0.98,且根毛侵染率与根内含菌量、发病率与根内含菌量的最大相关系数均出现在第6天,说明根肿菌早期侵染量直接影响白菜根肿病的发生程度。表明根内早期侵染是根肿菌致病的关键环节,通过水培法观测并结合实时荧光定量PCR检测可以评价品种的抗性,可作为根肿病抗性评价的一种方法。     

Root exudates of potato onion are involved in the suppression of clubroot in a Chinese cabbage-potato onion-Chinese cabbage crop rotation

Clubroot, caused by Plasmodiophora brassicae, has emerged as a serious disease threatening cruciferous crop production throughout the world. Crop rotation with non-host species is commonly practised to avoid clubroot, but it is not known whether rotation crops can control clubroot when the resting spores of P. brassicae remain unaffected. Pot experiments were performed to investigate the response of clubroot in Chinese cabbage to crop rotation with potato onion. The results showed that Chinese cabbage rotated with potato onion exhibited less clubroot disease than Chinese cabbage monoculture. Compared with residues from potato onion, the addition of root exudates from potato onion significantly decreased the disease incidence and index of clubroot (p ≤ 0.05). Potato onion root exudates decreased the number of secondary plasmodia of P. brassicae and the expression of the PRO1 gene of P. brassicae. These results suggest that root exudates from potato onion may play an important role in suppressing clubroot in a Chinese cabbage-potato onion-Chinese cabbage rotation system.     

Nature of resistance to Striga hermonthica (Del.) Benth. parasitism in some Sorghum vulgare (Pers.) cultivars

Ten Sorghum vulgare (Pers.) cultivars varying in tolerance to Striga hermonthica (Del.) Benth. parasitism were grown with or without Striga infection. Endodermal thickening, pericycle lignification and silica crystal deposition were studied microscopically and measured for infected and non-infected sorghum cultivars. Although differences in the root character measurements were statistically significant they were not closely related to the response of the plant to infection. Low stimulant producing cultivars showed low or medium root cell thickening. The cv. Framida had both low stimulant production and high root cell thickening and was the best of the tolerant cultivars. High stimulant producing, tolerant cultivars generally showed heavy or intermediate cell thickening. The high stimulant producing, susceptible cultivar Debaikri also showed intermediate root cell thickening.‘Antibiosis', measured by the content of phenolic compounds in the plant, was then studied. Varietal differences in quality and quantity of phenolic substances in the roots and shoots of sorghum cultivars infected or non-infected with Striga were observed. Infection increased total phenolic contents in both shoot and root extracts. Differences in the total phenolic content in the shoot of non-infected cultivars did not reflect tolerance to Striga infection. The total phenolic acid content of the root extracts was closely related to the response of the host plant to Striga infection, tolerant cultivars having greater total phenolic acid content than susceptible ones.     

Quantifying resistance to Plasmodiophora brassicae in Brassica hosts

Plasmodiophora brassicae causes clubroot of crucifers. A quantitative PCR (qPCR)‐based protocol was developed to measure P. brassicae DNA in the roots of susceptible, intermediately susceptible, intermediately resistant and resistant Brassica hosts, and the non‐host wheat, at 5, 10, 15, 20 and 42 days post‐inoculation (dpi). The final reaction of each plant genotype was recorded as an index of disease at 42 dpi. Plasmodiophora brassicae DNA showed an increase in susceptible and moderately resistant hosts from 5 to 42 dpi, in contrast to a decrease in a highly resistant host and the non‐host wheat over the same period. Index of disease was significantly positively correlated with the amount of P. brassicae DNA in the roots at 5, 15, 20 and 42 dpi in one experiment, and at 10, 15, 20 and 42 dpi in a repeated experiment. Significant positive correlations also existed between the amounts of P. brassicae DNA in the roots at 42 dpi and those at 5, 10, 15 and 20 dpi in one experiment, and those at 10, 15 and 20 dpi in a repeated experiment. The results generated by the qPCR assay were validated by microscopic examination of roots inoculated with P. brassicae. The qPCR‐based protocol developed in this study allows for the accurate quantification of P. brassicae DNA in host root tissues as early as 5 dpi, and may serve as a useful tool to evaluate pathogen proliferation and development in the roots.     

芸薹根肿菌次生游动孢子侵染致病分析

为明确芸薹根肿菌Plasmodiophora brassicae Woron.在其它寄主中是否广泛存在无性短循环及次生游动孢子的侵染致病性,以不结球白菜为寄主培养3批幼苗(G1、G2和G3),用休眠孢子悬浮液接种G1,被侵染的G1接种G2,被侵染的G2接种G3,采用离心管水培法研究其侵染致病性。结果显示,无性短循环研究中,G1、G2和G3根毛均被侵染,除G3并株接种侵染率为33.33%外,其它处理侵染率均在50.00%以上,根毛里有明显的游动孢子囊;次生游动孢子能侵染不结球白菜的皮层组织,致使不结球白菜发病形成明显的肿根;G1、G2和G3水培发病率为20.00%、15.00%和6.00%,砂培发病率为22.50%、18.75%和7.50%;G3肿根病理切片中可观察到休眠孢子。表明芸薹根肿菌侵染不结球白菜时,其生活史中存在无性短循环,次生游动孢子具有侵染致病作用。     

Tight regulation of allene oxide synthase (AOS) and allene oxide cyclase-3 (AOC3) promote <Emphasis Type="Italic">Arabidopsis</Emphasis> susceptibility to the root-knot nematode <Emphasis Type="Italic">Meloidogyne javanica</Emphasis>

Biosynthesis of the oxylipin jasmonic acid (JA) in Arabidopsis thaliana is catalyzed by a single allene oxide synthase (AOS)-encoding gene and four genes encoding four functional allene oxide cyclase (AOC) polypeptides (AOC1, AOC2, AOC3, and AOC4). To elucidate the biological activities of the JA pathway in regulating the plant defense response to plant-parasitic nematodes, transgenic lines carrying the GUS reporter gene under the control of individual AOC or AOS promoters were examined. Upon penetration by second-stage juveniles (J2 s), promoter activities of AOC1, AOC3 and AOC4 appeared in the root tip and root-elongation zone, with AOC3 demonstrating highest induction. At 5 days AOC3 activity continued to be highly pronounced in the stele and root cortex, associated with nematode invasion throughout gall initiation and maturation. AOS expression appeared 3 days postinfection and accompanied all later infection stages. Mutant lines were analyzed: disruption in AOS rendered plants more resistant to nematode infection, as reflected by the decreased number of females produced on this line; loss-of-function of AOC3 rendered plants more susceptible to nematode infection. Oxylipins derived from the 9- and 13-lipoxygenase pathways were assayed for their direct inhibitory activity toward M. javanica J2 s. Clear nematicidal activity of the bioactive 9- and 13-hydroperoxides was observed. Oxylipins produced by divinyl ether synthase, colneleic acid, colnelenic acid and ω5(Z)-etherolenic acid demonstrated strong inhibitory activity. These data, along with those of other assayed oxylipins, suggest that temporal and spatial fine tuning of the JA route allowing nematodes parasitism on plant host.     

Infection and colonization of peanut pods by Aspergillus parasiticus and the expression of the aflatoxin biosynthetic gene,nor-1, in infection hyphae

The aflatoxigenic fungi, Aspergillus flavus and A. parasiticus infect a wide variety of crops, all of which produce oil-rich seed. A histological study of the host–pathogen interaction between peanut,Arachis hyphogea , and A. parasiticus was performed in a system where peanuts remained attached to the plant and were inoculated without wounding. For infection studies, a genetically-tagged strain of A. parasiticus, G5, was engineered to harbor the β-glucuronidase (GUS) reporter gene under control of the nor-1 promoter from the aflatoxin biosynthetic pathway. There was a similar temporal pattern of aflatoxin B1 production and appearance of GUS activity in cultures ofA. parasiticus G5. This strain was used to follow infection and aflatoxin production during colonization of undamaged, drought-stressed peanuts. The fungus colonized all tissues of the peanut pod and appeared to gain ingress through the corky layer of the pericarp. Both intra- and inter-cellular colonization were observed. Fungal colonization of the cotyledons resulted in visible depletion of storage bodies within cells. Two morphologically distinct types of hyphae, wider hyphae and narrower hyphae, were seen throughout the pod tissues. Statistical analysis revealed that the narrower hyphae were significantly more likely to produce GUS activity than wider ones. GUS activity was found in hyphae infecting the pericarp, embryo and cotyledons indicating expression of aflatoxin biosynthetic genes in these tissues. Interestingly, GUS activity was not observed in the hyphae colonizing the testa.     

What can we learn from clubroots: alterations in host roots and hormone homeostasis caused by <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

The clubroot disease of cruciferous crops is caused by an obligate biotrophic protist, Plasmodiophora brassicae. The disease is characterized by the development of large root galls accompanied by changes in source-sink relations and the hormonal balance within the plant. Since the disease is difficult to control, it is of high economic interest to understand the events leading to gall formation. In this review we will give an overview on the current knowledge of changes brought about in the host root by this obligate biotrophic pathogen. Emphasis will be on the regulation of changes in plant hormone homeostasis, mainly auxins and cytokinins; the possible role of secondary metabolites, especially indole glucosinolates, in gall formation and auxin homeostasis will be discussed. Also, results from mutant analysis and microarrays using the model plant Arabidopsis thaliana are presented.     

Synthetic tetramer of a Phytophthora sojae-inducible fragment from soybean GmaSKTI36 promoter improves its pathogen induction activities

Using pathogen-induced promoters to control expression of the functional genes in transgenic plants may greatly increase the chances of boosting disease resistance. However, the number of the inducible promoters is limited. Here, we found that soybean GmaSKTI36 gene is strongly induced upon Phytophthora sojae infection. Functional analysis showed that its promoter could mediate rapid and strong induction of GUS expression upon pathogen infection in both Nicotiana benthamiana leaves and soybean hairy roots. Then, a 122 bp fragment that was critical to the activity was successfully identified by a progressive 5′ deletion analysis. Importantly, we found that a synthetic promoter by tetramerizing this fragment could confer strong P. sojae induction activities. Overall, the results suggested that the GmaSKTI36 promoter, the 122 bp fragment, and the synthetic promoter are potentially useful pathogen-inducible promoters.     

Suppression of clubroot (Plasmodiophora brassicae) development in Arabidopsis thaliana by the endophytic fungus Acremonium alternatum

This study investigated the ability of an endophytic fungus Acremonium alternatum to reduce clubroot formation in the model plant Arabidopsis thaliana, which is highly susceptible to Plasmodiophora brassicae . Quantitative PCR demonstrated that A. alternatum colonized the P. brassicae -infected roots and shoots of the host plant. When Arabidopsis plants were co-inoculated with P. brassicae and A. alternatum , gall formation was reduced as shown by the reduction of the disease index (DI) by up to 50% compared to plants only infected with P. brassicae, whereas the infection rate was lowered by about 20% only in several, but not all, experiments. Clubroot was similarly suppressed when plants were inoculated with autoclaved A. alternatum spores or spore extracts, showing that viable spores were not needed. However, A. alternatum spores did not inhibit P. brassicae resting spore germination. Compared to the normal root galls, the smaller root galls on A. alternatum -inoculated plants contained fewer resting spores of the clubroot pathogen. It was thus hypothesized that inoculation with A. alternatum delayed the development of P. brassicae . Using quantitative RT-PCR to monitor the expression of P. brassicae genes differentially expressed during the development of the disease, a delayed pathogen development was corroborated. Furthermore, greenhouse experiments identified a time window in which the endophyte had to be administered, where the latest effective time point was 5 days before inoculation with P. brassicae and the optimum treatment was to administer A. alternatum and P. brassicae at the same time. These results indicate that A. alternatum and perhaps similar endophytes could be useful for the management of clubroot disease.     

Biochemical factors involved in vetch resistance toOrobanche aegyptiaca

Experiments were performed to elucidate the biochemical nature of the differential response of resistantVicia atropurpureacv. Popany and susceptibleVicia sativacv. Yovel to the parasitic weedOrobanche aegyptiaca. Plant root material was obtained by growing vetch plants in association withOrobancheseeds on glass microfibre filter sheets inserted in polyethylene bags replenished by nutrient solution. Higher concentrations of bound phenolics, free phenolics and lignin, and higher activity of peroxidase were observed in cv. Popany roots as a result ofO. aegyptiacainfection than were found in cv. Yovel vetch. No differences in hydroxyproline-rich glycoproteins levels were detected between infected and non-infected roots of either vetch cultivar. These results suggest that the resistant vetch defense mechanism involves elevated induction of the phenylproponoid pathway uponOrobancheinfection, conferring mechanical and chemical barriers confronting the invading parasite.