<Emphasis Type="Italic">Fusarium sambucinum</Emphasis> isolate FS-94 induces resistance against fusarium wilt of tomato via activation and priming of a salicylic acid-dependent signaling system

The wheat rhizosphere-inhabiting nonpathogenic Fusarium sambucinum isolate FS-94 protected tomato from Fusarium wilt (F. oxysporum f. sp. lycopersici) in laboratory experiments. Seed soaking or immersion of seedling roots in a FS-94 spore suspension prior to inoculation with the pathogen delayed the appearance of wilt symptoms and significantly reduced disease severity in plants of a susceptible tomato cultivar. Quantification of fungal ergosterol in infected tomato showed that protection against wilt agent was related to limitation of the pathogen growth in plants exposed to FS-94. Incubation of tomato seedlings in a FS-94 spore suspension for 48 or 72 h led to plant protection and increased the salicylic acid (SA) concentration in their roots, suggesting that this isolate was involved in a plant-mediated mode of action and induced resistance. Soaking tomato seeds in the spore suspension did not induce SA accumulation in seedling roots, but nevertheless resulted in a significant reduction in wilt severity when the seedlings were challenged with the pathogen. In response to pathogen attack, the SA content in susceptible seedlings grown from FS-94-treated seeds started to increase within 1 day and remained elevated for 72 h. This suggests that F. sambucinum isolate FS-94 primed a SA-dependent signaling system in tomato.     

Arabidopsis ocp3 mutant reveals a mechanism linking ABA and JA to pathogen-induced callose deposition

In the present study, we evaluated the role of the defense-related gene OCP3 in callose deposition as a response to two necrotrophic fungal pathogens, Botrytis cinerea and Plectosphaerella cucumerina. ocp3 plants exhibited accelerated and intensified callose deposition in response to fungal infection associated with enhanced disease resistance to the two pathogens. A series of double mutant analyses showed potentiation of callose deposition and the heightened disease resistance phenotype in ocp3 plants required the plant hormone abscisic acid (ABA) and the PMR4 gene encoding a callose synthase. This finding was congruent with an observation that ocp3 plants exhibited increased ABA accumulation, and ABA was rapidly synthesized following fungal infection in wild-type plants. Furthermore, we determined that potentiation of callose deposition in ocp3 plants, including enhanced disease resistance, also required jasmonic acid (JA) recognition though a COI1 receptor, however JA was not required for basal callose deposition following fungal infection. In addition, potentiation of callose deposition in ocp3 plants appeared to follow a different mechanism than that proposed for callose β-amino-butyric acid (BABA)-induced resistance and priming, because ocp3 plants responded to BABA-induced priming for callose deposition and induced resistance of a magnitude similar to that observed in wild-type plants. Our results point to a model in which OCP3 represents a specific control point for callose deposition regulated by JA yet ultimately requiring ABA. These results provide new insights into the mechanism of callose deposition regulation in response to pathogen attack; however the complexities of the processes remain poorly understood.     

逆转座子Tos17的激活与水稻白叶枯病成株抗性的关系

为了研究水稻白叶枯病成株抗性是否与逆转子激活有关,运用SSAP (sequence-specific amplification polymorphism) 对成株抗性品种苗期和成株期接种白叶枯病原菌、清水接种及健康植株的基因组进行了逆转座子扫描。在筛选的约2000个逆转座子基因片段中,9个受苗期生长发育诱导激活,两个受成株期生长发育诱导激活,苗期和成株期各有3个受病原菌诱导激活。苗期生长发育诱导激活产生的逆转座子数目高于成株期,而病原菌诱导产生的逆转座子数目与成株期相当,表明水稻白叶枯病成株抗性可能与生长发育诱导的逆转座子激活相关。     

Histo-chemical changes induced by PGPR during induction of resistance in pearl millet against downy mildew disease

Plant growth-promoting rhizobacteria Bacillus pumilus strain INR-7 effectively induced downy mildew resistance in pearl millet. The histo-chemical analysis of B. pumilus INR-7 mediated systemic resistance showed that induced resistance is associated with the expression of hypersensitive response (HR), enhanced lignification, callose deposition, and hydrogen peroxide in addition to the increased expression of the defense enzymes β-1,3-glucanase, chitinase, phenylalanine ammonia lyase (PAL), peroxidase (POX), and polyphenol oxidase (PPO). There was rapid expression of HR in the resistant pearl millet as well as the susceptible seedlings induced by treatment with INR-7 after pathogen infection when compared to the susceptible seedlings, which expressed HR at later hours. Examination of inoculated pearl millet tissues by microscopy showed that lignin, callose, and hydrogen peroxide accumulated earlier and to higher levels in resistant and induced resistant seedlings. Accumulation of various defense enzymes was an immediate response to Sclerospora graminicola infection and preceded the development of induced resistance elicited by strain INR-7. Tissue print analysis showed that defense enzymes were found to be localized in the vascular bundles and revealed the visual difference in the expression pattern of β-1,3-glucanase, chitinase, PAL, POX, and PPO whose intensity varied among resistant, INR-7 treated, and susceptible pearl millet seedlings. This study clearly demonstrated that the differences between the responses, susceptible, INR-7 treated or resistant pearl millet seedlings recorded differences in the speed, intensity, and pattern of different histo-chemical responses to S. graminicola infection.     

Hypersensitive response, cell death and histochemical localisation of hydrogen peroxide in host and non-host seedlings infected with the downy mildew pathogen Sclerospora graminicola

Hypersensitive response, cell death and release of hydrogen peroxide as measures of host and non‐host defense mechanisms upon inoculation with the downy mildew pathogen Sclerospora graminicola were studied histochemically at the light microscopy level. The materials consisted of coleoptile tissues of the highly susceptible (cv. HB3), highly resistant (cv. IP18293) and induced resistant pearl millet host seedlings and non‐host sorghum (cv. SGMN10/8) and cotyledon of french bean (cv. S9). Resistance up to 80% protection against the downy mildew pathogen was induced in the highly susceptible HB3 cultivar of pearl millet by treating the seeds with 2% aqueous leaf extract of Datura metel for 3 h. Time course study with the pathogen inoculated highly resistant pearl millet cultivar revealed the appearance of hypersensitive response in 20% of seedlings as necrotic spots as early as 2 h after inoculation. In contrast, a similar reaction was observed in the highly susceptible pearl millet cultivar only 8 h after inoculation with the pathogen. In induced resistant seedlings, appearance of hypersensitive response was recorded 4 h after inoculation. Delayed hypersensitive response was observed in both the non‐host species at 10 h after inoculation. Hypersensitive response in the seedlings of the highly resistant pearl millet cultivar 24 h after inoculation showed 100% hypersensitive response, which was not observed in susceptible and non‐host species, although the induced resistant seedlings showed 90% hypersensitive response after that period of time. Cell death in the tissues of the test seedlings was also observed to change with time. Statistical analysis revealed that the tissues of highly resistant pearl millet seedlings required 2.9 h to attain 50% cell death. Tissues of induced resistant and highly susceptible pearl millet seedlings required 4.65 and 6.50 h respectively. In non‐hosts, 50% cell death was not recorded. Quantification of hydrogen peroxide in the tissue periplasmic spaces of the test seedlings revealed 2.94 h as the time required for 50% hydrogen peroxide accumulation in the tissues of highly resistant pearl millet seedlings. Tissues of induced resistant and highly susceptible pearl millet seedlings needed 3.76 and 5.5 h respectively. Fifty percent hydrogen peroxide localisation in non‐hosts could not be recorded. These results suggested the involvement of hydrogen peroxide, cell death and hypersensitive response in pearl millet host defense against S. graminicola.     

The identification of candidate genes associated with Pch2 eyespot resistance in wheat using cDNA-AFLP

Eyespot is a fungal disease of the stem base of cereal crops and causes lodging and the premature ripening of grain. Wheat cultivar Cappelle Desprez contains a highly durable eyespot resistance gene, Pch2 on the long arm of chromosome 7A. A cDNA-amplified fragment length polymorphism (AFLP) platform was used to identify genes differentially expressed between the eyespot susceptible variety Chinese Spring (CS) and the CS chromosome substitution line Cappelle Desprez 7A (CS/CD7A) which contains Pch2. Induced and constitutive gene expression was examined to compare differences between non-infected and plants infected with Oculimacula acuformis. Only 34 of approximately 4,700 cDNA-AFLP fragments were differentially expressed between CS and CS/CD7A. Clones were obtained for 29 fragments, of which four had homology to proteins involved with plant defence responses. Fourteen clones mapped to chromosome 7A and three of these mapped in the region of Pch2 making them putative candidates for involvement in eyespot resistance. Of particular importance are two fragments; 4CD7A8 and 19CD7A4, which have homology to an Oryza sativa putative callose synthase protein and a putative cereal cyst nematode NBS-LRR disease resistance protein (RCCN) respectively. Differential expression associated with Pch2 was examined by semi-quantitative RT-PCR. Of those genes tested, only four were differentially expressed at 14 days post inoculation. We therefore suggest that a majority of the differences in the cDNA-AFLP profiles are due to allelic polymorphisms between CS and CD alleles rather than differences in expression. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of cultivar and fungicides on stem-base pathogens, determined by quantitative PCR, and on diseases and yield of wheat

The polymerase chain reaction (PCR) was used to identify and quantify all fungal pathogens of wheat (Triticum aestivum) stem bases in nine field experiments at three locations in England. The main aim was to apply quantitative PCR to provide robust data on the efficacy of new fungicides against the individual components of the stem‐base disease complex. Cyprodinil most effectively controlled eyespot by decreasing both pathogens, Tapesia yallundae and T. acuformis (the most widespread species), and sometimes contributed to increased yields. Prochloraz controlled eyespot less consistently, its effectiveness dependent mainly on the presence of T. yallundae or on rainfall events soon after application. Azoxystrobin contributed to yield increases most consistently. Although it decreased sharp eyespot and its pathogen, Rhizoctonia cerealis, these effects were insufficient to account for much of the yield increases. The effects of fungicides on eyespot were sometimes greatest on the most susceptible cultivars. Amounts of Tapesia DNA were usually consistent with cultivar susceptibility ratings. The only pathogens of brown foot rot present in significant amounts were Microdochium nivale vars nivale and majus. They appeared not to affect yield or to respond greatly to fungicides. The susceptibility of cultivars to these pathogens was sometimes similar to their susceptibility to eyespot, suggesting that they may respond to the same host resistance genes or may in some cases be secondary colonisers of eyespot‐infected plants.     

Signalling requirements for Erwinia amylovora‐induced disease resistance,callose deposition and cell growth in the non‐host Arabidopsis thaliana

Erwinia amylovora is the causal agent of the fire blight disease in some plants of the Rosaceae family. The non‐host plant Arabidopsis serves as a powerful system for the dissection of mechanisms of resistance to E. amylovora. Although not yet known to mount gene‐for‐gene resistance to E. amylovora, we found that Arabidopsis activated strong defence signalling mediated by salicylic acid (SA), with kinetics and amplitude similar to that induced by the recognition of the bacterial effector avrRpm1 by the resistance protein RPM1. Genetic analysis further revealed that SA signalling, but not signalling mediated by ethylene (ET) and jasmonic acid (JA), is required for E. amylovora resistance. Erwinia amylovora induces massive callose deposition on infected leaves, which is independent of SA, ET and JA signalling and is necessary for E. amylovora resistance in Arabidopsis. We also observed tumour‐like growths on E. amylovora‐infected Arabidopsis leaves, which contain enlarged mesophyll cells with increased DNA content and are probably a result of endoreplication. The formation of such growths is largely independent of SA signalling and some E. amylovora effectors. Together, our data reveal signalling requirements for E. amylovora‐induced disease resistance, callose deposition and cell fate change in the non‐host plant Arabidopsis. Knowledge from this study could facilitate a better understanding of the mechanisms of host defence against E. amylovora and eventually improve host resistance to the pathogen.     

Fusarium oxysporum f. sp. psidii as a Pathogen Causing Wilt of Guava in Varanasi District, India

A serious wilt disease of guava has been observed in the Varanasi district of eastern Uttar Pradesh of India. The causal organism has been identified as Fusarium oxysporum f. sp. psidii. Pathogenicity tests were performed in pot experiments to confirm the causal agent of the disease. Infected plants developed chlorosis followed by wilting of entire seedlings and leaf abscission. Histopathological studies showed the presence of hyphae in xylem vessels of roots of the wilted seedlings and when sections of such roots were transferred to potato dextrose agar medium, this pathogen grew in culture.     

Salicylic acid is important for basal defense of Solanum tuberosum against Phytophthora infestans

The importance of the signaling compound salicylic acid for basal defense of potato (Solanum tuberosum L. cv. Désirée) against Phytophthora infestans, the causal agent of late blight disease, was assessed using transgenic NahG potato plants which are unable to accumulate salicylic acid. Although the size of lesions caused by P. infestans was not significantly different in wild-type and transgenic NahG plants, real-time polymerase chain reaction analyses revealed a drastic enhancement of pathogen growth in potato plants depleted of salicylic acid. Increased susceptibility of NahG plants correlated with compromised callose formation and reduced early defense gene expression. NahG plants pretreated with the salicylic acid analog 2,6-dichloro-isonicotinic acid allowed pathogen growth to a similar extent as did wild-type plants, indicating that salicylic acid is an important compound required for basal defense of potato against P. infestans.     

Comparative proteomic analysis of methyl jasmonate‐induced defense responses in different rice cultivars

Jasmonate is an important endogenous chemical signal that plays a role in modulation of plant defense responses. To understand its mechanisms in regulation of rice resistance against the fungal pathogen Magnaporthe oryzae, comparative phenotype and proteomic analyses were undertaken using two near‐isogenic cultivars with different levels of disease resistance. Methyl‐jasmonate (MeJA) treatment significantly enhanced the resistance against M. oryzae in both cultivars but the treated resistant cultivar maintained a higher level of resistance than the same treated susceptible cultivars. Proteomic analysis revealed 26 and 16 MeJA‐modulated proteins in resistant and susceptible cultivars, respectively, and both cultivars shared a common set of 13 proteins. Cumulatively, a total of 29 unique MeJA‐influenced proteins were identified with many of them known to be associated with plant defense response and ROS accumulation. Consistent with the findings of proteomic analysis, MeJA treatment increased ROS accumulation in both cultivars with the resistant cultivar showing higher levels of ROS production and cell membrane damage than the susceptible cultivar. Taken together, our data add a new insight into the mechanisms of overall MeJA‐induced rice defense response and provide a molecular basis of using MeJA to enhance fungal disease resistance in resistant and susceptible rice cultivars.     

Interplay between JA, SA and ABA signalling during basal and induced resistance against Pseudomonas syringae and Alternaria brassicicola

We have examined the role of the callose synthase PMR4 in basal resistance and β-aminobutyric acid-induced resistance (BABA-IR) of Arabidopsis thaliana against the hemi-biotrophic pathogen Pseudomonas syringae and the necrotrophic pathogen Alternaria brassicicola . Compared to wild-type plants, the pmr4-1 mutant displayed enhanced basal resistance against P. syringae , which correlated with constitutive expression of the PR-1 gene. Treating the pmr4-1 mutant with BABA boosted the already elevated levels of PR-1 gene expression, and further increased the level of resistance. Hence, BABA-IR against P. syringae does not require PMR4-derived callose. Conversely, pmr4-1 plants showed enhanced susceptibility to A. brassicicola , and failed to show BABA-IR. Wild-type plants showing BABA-IR against A. brassicicola produced increased levels of JA. The pmr4-1 mutant produced less JA upon A. brassicicola infection than the wild-type. Blocking SA accumulation in pmr4-1 restored basal resistance, but not BABA-IR against A. brassicicola . This suggests that the mutant's enhanced susceptibility to A. brassicicola is caused by SA-mediated suppression of JA, whereas the lack of BABA-IR is caused by its inability to produce callose. A. brassicicola infection suppressed ABA accumulation. Pre-treatment with BABA antagonized this ABA accumulation, and concurrently potentiated expression of the ABA-responsive ABI1 gene. Hence, BABA prevents pathogen-induced suppression of ABA accumulation, and sensitizes the tissue to ABA, causing augmented deposition of PMR4-derived callose.     

Induced resistance activated by a culture filtrate derived from an avirulent pathogen as a mechanism of biological control of anthracnose in strawberry

In a previous report, it was described that strawberry plants pre-treated with an avirulent isolate of Colletotrichum fragariae (M23) acquired resistance to a virulent isolate of Colletotrichum acutatum (M11) causing anthracnose. In this report we present evidence that the eliciting activity can be found not only in conidial extracts but in culture supernatants of the avirulent pathogen as well. Plants of the cv. Pájaro treated with the culture filtrate (CF) derived from M23, 3 days prior to the inoculation with M11 showed significantly reduced disease severity as compared to control plants and the disease was completely suppressed when plants were pre-treated 7 days before the challenge inoculation with M11. The same effect was achieved when a single leaf was sprayed with CF, suggesting that the resistance acquired is systemic. Control treatments showed that none of the active extracts inhibited the growth of the virulent pathogen, indicating that the protection effect was due to the induction of a defense response. The latter was confirmed by the accumulation of reactive oxygen species (e.g. hydrogen peroxide, superoxide anion) and the deposition of lignin and callose, usually associated to plant defense, after the CF treatment. Experiments carried out with other strawberry cultivars treated with CF showed that also protected them against different virulent isolates, suggesting that the response observed is cultivar-nonspecific. These outcomes indicate that the protection against anthracnose in strawberry involves a phenomenon of induced resistance (IR) by action of defense-eliciting molecules produced by M23.     

Cell membrane integrity,callose accumulation,and root growth in aluminum-stressed sorghum seedlings

Aluminum stress usually reduces plant root growth due to the accumulation of Al in specific zones of the root apex. The objectives of this study were to determine the localization of Al in the root apex of Sorghum bicolor (L.) Moech. and its effects on membrane integrity, callose accumulation, and root growth in selected cultivars. Seedlings were grown in a nutrient solution containing 0, 27, or 39 μM Al³⁺ for 24, 48, and 120 h. The Al stress significantly reduced root growth, especially after 48 and 120 h of exposure. A higher Al accumulation, determined by fluorescence microscopy after staining with a Morin dye, occurred in the root extension zone of the sensitive cultivar than in the tolerant cultivar. The membrane damage and callose accumulation were also higher in the sensitive than resistant cultivar. It was concluded that the Al stress significantly reduced root growth through the accumulation of Al in the root extension zone, callose accumulation, and impairment of plasma membrane integrity.     

Simulation of fungal-mediated cell death by fumonisin B1 and selection of fumonisin B1-resistant (fbr) Arabidopsis mutants

Fumonisin B1 (FB1), a programmed cell death-eliciting toxin produced by the necrotrophic fungal plant pathogen Fusarium moniliforme, was used to simulate pathogen infection in Arabidopsis. Plants infiltrated with 10 microM FB1 and seedlings transferred to agar media containing 1 microM FB1 develop lesions reminiscent of the hypersensitive response, including generation of reactive oxygen intermediates, deposition of phenolic compounds and callose, accumulation of phytoalexin, and expression of pathogenesis-related (PR) genes. Arabidopsis FB1-resistant (fbr) mutants were selected directly by sowing seeds on agar containing 1 microM FB1, on which wild-type seedlings fail to develop. Two mutants chosen for further analyses, fbr1 and fbr2, had altered PR gene expression in response to FB1. fbr1 and fbr2 do not exhibit differential resistance to the avirulent bacterial pathogen Pseudomonas syringae pv maculicola (ES4326) expressing the avirulence gene avrRpt2 but do display enhanced resistance to a virulent isogenic strain that lacks the avirulence gene. Our results demonstrate the utility of FB1 for high-throughput isolation of Arabidopsis defense-related mutants and suggest that pathogen-elicited programmed cell death of host cells may be an important feature of compatible plant-pathogen interactions.     

Expression of callose synthase genes and its connection with <Emphasis Type="Italic">Npr1</Emphasis> signaling pathway during pathogen infection

Callose synthesis occurs at specific stages of plant cell wall development in all cell types, and in response to pathogen attack, wounding and physiological stresses. We determined the expression pattern of "upstream regulatory sequence" of 12 Arabidopsis callose synthase genes (CalS1-12) genes and demonstrated that different callose synthases are expressed specifically in different tissues during plant development. That multiple CalS genes are expressed in the same cell type suggests the possibility that CalS complex may be constituted by heteromeric subunits. Five CalS genes were induced by pathogen (Hyaloperonospora arabidopsis, previously known as Peronospora parasitica, the causal agent of downy mildew) or salicylic acid (SA), while the other seven CalS genes were not affected by these treatments. Among the genes that are induced, CalS1 and CalS12 showed the highest responses. In Arabidopsis npr1 mutant, impaired in response of pathogenesis related (PR) genes to SA, the induction of CalS1 and CalS12 genes by the SA or pathogen treatments was significantly reduced. The patterns of expression of the other three CalS genes were not changed significantly in the npr1 mutant. These results suggest that the high induction observed of CalS1 and CalS12 is Npr1 dependent while the weak induction of five CalS genes is Npr1 independent. In a T-DNA knockout mutant of CalS12, callose encasement around the haustoria on the infected leaves was reduced and the mutant was found to be more resistant to downy mildew as compared to the wild type plants.