Arginase activity in Arabidopsis thaliana infected with Heterodera schachtii

The activity of arginase (ARGAH), which results in ornithine and urea production, is important for nitrogen metabolism in all organisms as well as for defence responses. The second‐stage juveniles of the cyst‐forming nematode Heterodera schachtii penetrate roots and induce the formation of a permanent feeding site. To determine whether infection with H. schachtii causes the induction of arginase, the expression was studied in Arabidopsis roots and shoots at the day of inoculation and at 3, 7 and 15 days post‐inoculation (dpi). Parasite infection caused a strong decrease of root arginase activity and ARGAH1 gene expression, which persisted over the entire examination period. In shoots, the mRNA expression of ARGAH2 increased at 3 and 7 dpi, but the enzymatic activity was significantly enhanced only at 3 dpi. Thus, while arginase down‐regulation occurs in roots, which is apparently due to the presence of nematode effectors, in shoots the activity is only transiently up‐regulated despite persistently high gene expression. As oxidative stress is possible during nematode infection, the activity and gene expression of glutathione reductase, a marker of the redox equilibrium, were estimated and found to be significantly enhanced at 7 dpi in shoots of infected plants. The level of proline, an amino acid known for its ability to scavenge free radicals, was increased 60‐fold. The results suggest that the disruption of redox homeostasis, as reflected by increased proline level and glutathione reductase expression and activity, accompanies changes in arginine metabolism in the shoots, indicating systemic changes induced by nematode infection.     

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.     

橡胶树白粉菌侵染过程转录组学分析

为明确橡胶树白粉菌Erysiphe quercicola参与致病过程相关基因的表达情况，基于RNA-Seq测序技术对橡胶树白粉菌侵染过程进行转录调控研究，通过对病原菌孢子（0 h）及3个侵染时期（接种1、3和30 d）的转录组进行比较，筛选差异表达基因并对其进行功能注释分析，同时对不同侵染阶段的基因表达趋势进行聚类分析。结果表明，相比于病原菌孢子，3个侵染时期（接种后1、3和30 d）分别有198、458和27个差异表达基因。基因功能富集分析发现氧化还原酶相关基因在侵染1 d阶段显著富集，可能参与病原菌侵染前期对活性氧的防御。基因表达趋势聚类分析显示不同侵染阶段的基因共分为51种表达类型，其中编码候选效应蛋白基因集中分布在侵染1 d后上调表达的6个类型当中。表明橡胶树白粉菌侵染过程相关基因具有明显的功能倾向性和表达趋势特征。     

<Emphasis Type="Italic">In planta</Emphasis> multiplication and graft transmission of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter asiaticus’ revealed by Real-Time PCR

The bacterium Candidatus Liberibacter asiaticus (CLas) is associated with huanglongbing (HLB) in citrus in many countries. Despite the fact that many characteristics of the disease are known, the rate of multiplication of the bacterium within an infected tree is still poorly understood. To study this feature, we used the quantitative Real-Time polymerase chain reaction (Q-PCR) assay to follow and to quantify the multiplication of CLas in grafted infected young sweet orange plants. The rate of infection by grafting reached 100% at 120 days post-inoculation (dpi) showing that grafting could easily transmit CLas. A well-adjusted linear regression equation describing the bacterial growth in planta was obtained independently with measurements taken using repeated sampling in the same plant or different plants through the analysed period. The bacterial population, measured as copy number (CN) of the 16S rDNA target gene g⁻¹ of tissue, increased 10,000 times from 10³ at 30 dpi to approximately 10⁸ CN at 240 dpi indicating that CLas multiplication was fastest in young citrus plants. We observed a direct relationship between the concentration of pathogen and the expression of symptoms. Yellowed leaves or shoots, are commonly the first observed symptom of HLB, and were present in trees with a low amount of bacteria (10⁵ CN g⁻¹). Blotchy mottle symptoms were observed in trees with 10⁷ CN g⁻¹ of bacteria after 180 dpi. Buds taken from infected, but non-symptomatic branches were grafted on Rangpur lime and resulted in transmission rates ranging from 10 to 60%.     

Spatiotemporal changes in fungal growth and host responses of six yellow rust resistant near-isogenic lines of wheat

The objective of the present study was to investigate to what extent the macroscopic phenotype of incompatible host–pathogen interactions reflects differences in fungal development and host responses at the histological level. This was done by conventional and advanced microscopic analysis of six wheat near-isogenic lines differing by individual R genes and inoculated with an avirulent isolate of Puccinia striiformis. Wheat line AvocetYr15 had the lowest macroscopic infection type (IT) 0–1, in which fungal growth was stopped at early stages due to extensive expression of a hypersensitive response (HR) at all time points (4, 8 and 16 days post-inoculation, dpi) without any sign of haustorial bodies. AvocetYr5 and AvocetYr1 had IT 1, in which most fungal colonies developed secondary hyphae. Many colonies were encased by HR at 16 dpi, more extensively in AvocetYr1 than AvocetYr5. In AvocetYr6 (IT 2), HR was expressed after the formation of secondary hyphae at 4 dpi, after which fungal growth and HR increased simultaneously until most colonies became encased by HR. AvocetYr27 (IT 2–3) and AvocetYr17 (IT 4–5) showed similar fungal growth and HR at 4 dpi, where HR was only weakly expressed in a few host cells. Encasement of secondary and runner hyphae increased significantly in AvocetYr27, but at 16 dpi, HR was often circumvented by large, intermingled fungal colonies in both lines. No resistance responses were observed in Avocet S (susceptible control). The very different histological patterns conferred by the six R genes suggests differences in the timing of the host–pathogen recognition and onset of host defence pathways.     

A histological assessment of the infection strategy of Exserohilum turcicum in maize

Northern leaf blight is a lethal foliar disease of maize caused by the fungus Exserohilum turcicum. The aim of this study was to elucidate the infection strategy of the fungus in maize leaves using modern microscopy techniques and to understand better the hemibiotrophic lifestyle of E. turcicum. Leaf samples were collected from inoculated B73 maize plants at 1, 4, 9, 11, 14 and 18 days post-inoculation (dpi). Samples were prepared according to standard microscopy procedures and analysed using light microscopy as well as scanning (SEM) and transmission electron microscopy (TEM). Microscopic observations were preceded by macroscopic observations for each time point. The fungus penetrated the leaf epidermal cells at 1 dpi and the disease was characterized by chlorotic leaf flecks. At 4 dpi the chlorotic flecks enlarged to form spots, and at 9 dpi hyphae were seen in the epidermal cells surrounding the infection site. At 11 dpi lesions started to form on the leaves and SEM revealed the presence of hyphae in the vascular bundles. At 14 dpi the xylem was almost completely blocked by hyphal growth. Hyphae spread into the adjacent bundle sheath cells causing cellular damage, characterized by plasmolysis, at 18 dpi and conidiophores formed through the stomata. Morphologically, lesions started to enlarge and coalesce leading to wilting of leaves. This study provides an updated, detailed view of the infection strategy of E. turcicum in maize and supports previous findings that E. turcicum follows a hemibiotrophic lifestyle.     

Effect of temperature on resistance to Potato virus Y in potato cultivars carrying the resistance gene Rychc

The effect of cultivation temperatures on the resistance reaction to three Potato virus Y strains (PVY^O, PVY^N and PVY^NTN) in potato cultivars carrying Ry_chc was examined. When potato plants carrying Ry_chc were cultivated at 22 °C, a few small necrotic spots developed on inoculated leaves by 5 days after mechanical inoculation (dpi), and systemic infection of a few symptomless plants was confirmed at 28 dpi by IC‐RT‐PCR. At 28 °C, distinct necrotic spots developed on inoculated leaves by 5 dpi, and systemic symptoms occasionally appeared at 28 dpi. Thus, high temperature weakens Ry_chc‐conferred resistance. However, the incidence of systemic infection and the titre of virus in resistant cultivars at 28 °C were lower than in a susceptible cultivar. In graft inoculation under high summer temperatures, some plants developed necrosis on the leaves and stem, but PVY was barely detected by RT‐PCR in leaves on potato carrying Ry_chc. When seedlings from progeny tubers of plants that were inoculated with PVY and grown in a greenhouse at >30 °C in the daytime were examined by ELISA and IC‐RT‐PCR, PVY was not detected in cultivars carrying Ry_chc. These results show that Ry_chc confers an extreme resistance to PVY strains occurring in Japan.     

Effects of panicle development stage and temperature on rice panicle blast infection by Magnaporthe oryzae and visualization of its infection process

Panicle blast, caused by the fungus Magnaporthe oryzae (syn. Pyricularia oryzae), directly contributes to yield loss in the field. The effects of panicle development stage and temperature on panicle blast were studied and the infection process of M. oryzae in panicles was visualized. Rice panicles at different development stages from three rice cultivars were inoculated with a conidial suspension in vitro. The rice cultivar Lijiangxintuanheigu was highly susceptible to panicle blast at 5 days postinoculation (dpi) when the pulvinus distance was 15–20 cm. Nanjing 9108 was moderately susceptible to panicle blast when the pulvinus distance was 8–10 cm, but Yliangyou 800 was resistant. The effect of temperature on panicle blast was determined under 22–35 °C temperature treatments. Inoculated panicles placed at temperatures of 28 and 30 °C showed the highest lesion grade based on lesion length at 5 dpi. The infection process of M. oryzae in rice panicles was observed by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). M. oryzae initially formed the appressorium to invade through the epidermis of rice panicles at 24 hours postinoculation (hpi). As the disease progressed, the invasive hyphae formed dense mycelial networks in the inner parenchyma cells at 60 hpi. Our results will contribute to the understanding of panicle development stage and temperature effects on panicle blast and improve resistance evaluation methods. Additionally, visualization of the infection process by CLSM and TEM are valuable methods to observe M. oryzae invasive hyphae inside rice panicle cells.     

Influence of an m-type thioredoxin in maize on potyviral infection

Expression of many host genes can be altered during virus infection. In a previous study of sugarcane mosaic virus (SCMV) infection in maize (Zea mays), we observed that expression of ZmTrm2, a gene encoding thioredoxin m, was up-regulated at about 10 days post-inoculation (dpi). In this present study we determined that ZmTrm2 silencing in maize by virus-induced gene silencing significantly enhanced systemic SCMV infection. In contrast transient over-expression of ZmTrm2 in maize protoplasts inhibited accumulation of SCMV viral RNA. Furthermore, we found that in inoculated Nicotiana tabacum leaves transient expression of ZmTrm2 inhibited accumulation of the RNA of tobacco vein-banding mosaic virus (TVBMV), a potyvirus infecting dicotyledonous plants. Interestingly in ZmTrm2 transiently expressed N. tabacum leaves, we detected by semi-quantitative RT-PCR a reduced level of the mRNA of class I beta-1, 3-glucanase (GluI), a protein known to have a role in cell wall callose deposition and viral movement. Our data indicate that the maize ZmTrm2 plays an inhibitory role during infection of plants by SCMV and TVBMV.     

Detached leaf inoculation of germplasm for rapid screening of resistance to citrus canker and citrus bacterial spot

A detached leaf protocol for rapid screening of germplasm for resistance to citrus canker (Xanthomonas citri subsp. citri, Xcc) and citrus bacterial spot (Xanthomonas alfalfae subsp. citrumelonis, Xac) was developed to evaluate limited quantities of leaf material. Bacterial inocula of Xcc or Xac at 10⁴, 10⁵, or 10⁸ cfu ml⁻¹ were injection-infiltrated into the abaxial surface of disinfested, immature leaves of susceptible and resistant genotypes. Inoculated detached leaves were placed on the surface of 0.5% water agar plates and incubated at 28°C under a 12 h photoperiod. Likewise, inocula were infiltrated into attached leaves of greenhouse plants. At high inoculum concentrations of Xcc or Xac (10⁸ cfu ml⁻¹), resistant cultivars of kumquat developed a hypersensitive-like reaction within 3 days post inoculation (dpi). At 10⁵ cfu ml⁻¹, populations 14 dpi were <10⁴ per inoculation site. In canker-susceptible Citrus spp. (‘Duncan’ grapefruit and ‘Rough’ lemon), water-soaked areas occurred by 3 dpi and typical canker lesions developed by 7 to14 dpi. Concentration of Xcc recovered from inoculation sites was approximately 10⁵ cfu ml⁻¹ by 14 dpi. In citrus bacterial spot-susceptible citrus (‘Swingle’ citrumelo and grapefruit), symptoms developed within 7 dpi. Populations of Xac after inoculation at 10⁵ cfu ml⁻¹ were comparable to Xcc in susceptible hosts 14 dpi (>10⁵). The detached leaf assay is useful for the characterization and differentiation of lesion phenotype for each Xanthomonas pathogen permitting rapid screening of germplasm resistance based on the quantification of number of lesions and bacterial concentration.     

Evidence of strong gene flow among French populations of the carrot cyst nematode Heterodera carotae

The recent ban of the most efficient chemical nematicides has left growers without methods for controlling the carrot cyst nematode Heterodera carotae. This phytoparasitic nematode species has a very narrow host range and causes severe crop losses in the main carrot-growing regions worldwide. The development of alternative means of management of H. carotae is thus essential, and knowledge is required about the adaptive abilities of H. carotae, which mainly depend on gene flow among populations. The goal of this study was to describe the genetic structure of H. carotae populations at the spatial scale of the main infested French carrot-producing region, i.e. Lower Normandy, and to disentangle the causes of the heterozygote deficit in this polyvoltine species. Microsatellite genotyping of populations collected at both the plant and field scales showed that: (i) the heterozygote deficit is mainly due to substructure; and (ii) there is strong gene flow among populations, leading to low F_ST and to no clear genetic structure at the spatial scale explored here. Soil transport through both agricultural machinery and the transport of leek seedlings is probably responsible for the very strong H. carotae migration among fields and production areas. Measures should be considered to limit the passive spread of H. carotae.     

Reaction of three potato cultivars to potato virus S and alteration of miRNAs and mRNA targets in the resistant genotypes

Reactions of three Polish potato cultivars to potato virus S (PVS) were investigated at 22°C. Cultivars Tajfun and Tonacja exhibited partial resistance with systemic infection detected in some inoculated plants; cultivar Bryza was susceptible to PVS with systemic infection detected in all inoculated plants. The virus was not detectable by ELISA at 23 days postinoculation (dpi) but was detected after 40 dpi. Infection rate and viral accumulation were significantly lower in Tonacja and Tajfun than in Bryza, but no statistically significant difference between Tajfun and Tonacja was detected. Both susceptible and resistant genotypes displayed various, either common or cultivar-specific, symptoms. Delayed systemic infection at 56 dpi was observed in some cases in Tonacja and Tajfun. Resistance-related alteration of a set of miRNAs and mRNA targets in the tested cultivars in response to PVS at 22°C exhibited inter- and intracultivar variability. The majority of tested genes were altered only in the partially resistant Tajfun and Tonacja but not in the susceptible Bryza. Enhanced expression of AGO1-2, DCL1, stu-miR482 and its target Gpa2 was observed in Tonacja and plants of Tajfun in which PVS was detected, with the highest induction of Gpa2 in Tajfun (30.2-fold). However, their expression remained unchanged or decreased in plants of Tajfun in which PVS was undetected. Increased expression of stu-miR168a and stu-miR172e was observed in Tonacja and the PVS-undetected plants of Tajfun, respectively, but not in the PVS-detected plants of Tajfun. This is the first report on cultivar-specific alteration of miRNA in a potato–PVS resistance interaction.     

Up‐regulation of PR1 and less disruption of hormone and sucrose metabolism in roots is associated with lower susceptibility to ‘Candidatus Liberibacter asiaticus’

Huanglongbing (HLB), caused by ‘Candidatus Liberibacter asiaticus’ (Las), is a devastating disease of citrus trees in Florida. Previous work showed that the rootstock cultivar Cleopatra mandarin (Citrus reticulata) has a higher population of Las in roots than Swingle citrumelo (C. paradisi × Poncirus trifoliata). Las reduced fibrous root biomass and sucrose content in Cleopatra mandarin more than in Swingle citrumelo. To understand the mechanisms for susceptibility to Las infection, sucrose and hormone metabolism status were evaluated in Cleopatra mandarin and Swingle citrumelo. In fibrous roots of Cleopatra mandarin, higher expression of genes related to sucrose cleavage was consistent with lower sucrose content compared to noninoculated seedlings at 5 weeks post‐root trimming (wpt). In fibrous roots of Swingle citrumelo, both sucrose content and gene expression related to sucrose cleavage were less disrupted by Las infection compared to Cleopatra mandarin at 5 wpt. Genes associated with salicylic acid (SA), ethylene (ET) and abscisic acid (ABA) synthesis, and ABA signalling, phospholipases D (PLD), and phospholipase A2 (PLA2) were activated by Las infection at 5 wpt in Cleopatra mandarin. Expression of downstream effectors of SA, i.e. NPR1, WRKY70 and PR1, did not change in Cleopatra mandarin, suggesting inhibition of the response to SA by the elevation of ABA, ET and PLD. In contrast, the up‐regulation of PR1, lower response of sucrose metabolism genes and down‐regulation of biosynthesis of phytohormones indicates that Swingle citrumelo activates a more effective defence against this biotrophic pathogen than Cleopatra mandarin.     

Systemic accumulation of novel proteins in the apoplast of the leaves of potato plants following root invasion by the cyst-nematode Globodera rostochiensis

Systemic changes in gene expression have been analysed in potato plants following root infection with the cyst nematode Globodera rostochiensis. Root invasion by the pathogen induced major changes to occur in the composition of proteins extracted in intercellular fluid from the leaf apoplastic space. The changes involved the disappearance of innate leaf proteins and the accumulation of novel gene products. Analysis by SDS-PAGE indicated that the leaf polypeptides induced on nematode invasion are not cultivar or nematode pathotype-specific. Aspirin treatment of leaves induced a similar pattern to that induced systemically by nematodes, but several gene products were specific for the latter method of induction. In particular, two abundant [¹²⁵I]-Con A binding polypeptides of high M_r accumulated in intercellular fluid only on aspirin treatment. The results are discussed in the context of the role of the extracellular leaf proteins and their modulation by systemic signals.     

ROS and NO production in compatible and incompatible tomato-<Emphasis Type="Italic">Meloidogyne incognita</Emphasis> interactions

Nitric oxide (NO) has been shown to be an essential regulatory molecule in plant response to pathogen infection in synergy with reactive oxygen species (ROS). At the present, nothing is known about the role of NO in disease resistance to nematode infection. We used a resistant tomato cultivar with different sensitivity to avirulent and virulent populations of the root-knot nematode Meloidogyne incognita to investigate the key components involved in oxidative and nitrosative metabolism. We analyzed the superoxide radical production, hydrogen peroxide content, and nitric oxide synthase (NOS)-like and nitrate reductase activities, as potential sources of NO. A rapid NO accumulation and ROS production were found at 12 h after infection in compatible and incompatible tomato-nematode interactions, whereas the amount of NO and ROS gave different results 24 and 48 h after infection amongst compatible and incompatible interactions. NOS-like arginine-dependent enzyme rather than nitrate reductase was the main source of NO production, and NOS-like activity increased substantially in the incompatible interaction. We can envisage a functional overlap of both NO and ROS in tomato defence response to nematode invasion, NO and H₂O₂ cooperating in triggering hypersensitive cell death. Therefore, NO and ROS are key molecules which may help to orchestrate events following nematode challenge, and which may influence the host cellular metabolism.