Induction of a defense response in strawberry mediated by an avirulent strain of <Emphasis Type="Italic">Colletotrichum</Emphasis>

In the strawberry crop area of Tucumán (north-west Argentina) the three species of Colletotrichum causing anthracnose disease (C. acutatum, C. fragariae and C. gloeosporioides) were detected. Among all isolates characterized, one of them identified as C. acutatum (M11) and another as C. fragariae (F7) were selected due to their conspicuous interaction with the strawberry cultivar Pájaro. Whereas isolate M11 produced a strong compatible interaction in cv. Pájaro with clear disease symptoms (DSR = 5.0), the isolate F7 brought about a typical incompatible interaction (DSR = 1.0). When plants of cv. Pájaro were inoculated with F7 prior to the inoculation with M11, the former avirulent strain prevented the growth of the latter virulent pathogen. Experimental evidence indicated that the time elapsed between the first inoculation with the avirulent pathogen and the second inoculation with the virulent one was crucial to inhibit the growth of the latter. The growth of F7 on the plant without provoking damage and the fact that there was no in vitro antagonistic effect between the pathogens, suggests that the avirulent strain triggers a plant defensive response against M11. The defense response was further confirmed by the detection of an early oxidative burst occurring within 4 h after the first inoculation and by the observation of anatomical changes associated with defense mechanisms that lasted 50 days after the inoculation with F7. Results obtained support the hypothesis that the plant resistance against the virulent strain M11 is elicited by one or more diffusible(s) compound(s) produced by the avirulent strain F7.     

Programmed cell death pathways induced by early plant‐virus infection are determined by isolate virulence and stage of infection

Programmed cell death (PCD) pathways caused by Turnip mosaic virus (TuMV) infection before symptom appearance were studied by light microscopy and electrolyte leakage following sap inoculation of Brassica carinata (Ethiopian mustard) TZ‐SMN‐44‐6 plants. Leaf responses to inoculation with avirulent (TuMV‐avir) and virulent (TuMV‐vir) isolates, and mock‐inoculation, were compared at 2, 20 and 52 h after inoculation (hai). The phenotypes induced were localized resistance (TuMV‐avir) and systemic susceptibility (TuMV‐vir). No visible TuMV symptoms were recorded in any inoculated plants during the 2–52 hai sampling period, but appeared as chlorotic spots in inoculated leaves at 5 days after inoculation. With TuMV‐vir alone, they were followed by systemic infection (mosaic). Dead cell number, deformation, percentage area and percentage integrated intensity, and conductivity of electrolyte leakage data, were analysed to examine their possible roles in stimulating cell death pathways. At 2 hai, dead cell number and percentage area were significantly greater for TuMV‐avir than TuMV‐vir infection or mock‐inoculation. Overall, isolate TuMV‐vir caused significantly greater cell deformation than TuMV‐avir, whereas wounding by mock‐inoculation had negligible effects. By 52 hai, isolate TuMV‐avir caused significantly greater electrolyte leakage than isolate TuMV‐vir or mock‐inoculation. This suggests both isolates triggered morphological changes consistent with apoptotic‐like PCD and necrosis‐like PCD that depended upon isolate virulence and stage of infection, respectively. These findings highlight how quantification of dead cell deformation and electrolyte leakage offer a new understanding of compatible and incompatible plant responses to early virus infection in plants.     

Differential necrotic lesion formation in soybean cultivars in response to soybean mosaic virus

In this study, we examined the necrosis phenotype on leaves of two cultivars of soybean (ZheA8901 and Nannong1138-2) that show varying level of resistance to soybean mosaic virus (SMV). The necrotic symptoms seen on inoculated and systemic leaves of soybean cultivar ZheA8901 were reminiscent of programmed cell death (PCD). The cell death phenotypes were evaluated using the TUNEL method, quantification of hydrogen peroxide (H₂O₂) and salicylic acid, callose production, as well as by monitoring expression of defence genes GmPR-1 and GmNPR1. Our results show that SMV inoculation induced PCD on ZheA8901 is associated with rapid increase in H₂O₂, increased SA and callose accumulation and higher defence gene expression.     

Age-related resistance to Pseudomonas syringae pv. tomato is associated with the transition to flowering in Arabidopsis and is effective against Peronospora parasitica

As plants mature it has been observed that some become more resistant to normally virulent pathogens. The ability to manifest the Age-Related Resistance (ARR) response in Arabidopsis to Pseudomonas syringae pathovars tomato (Pst) coincided with the transition to flowering in plants both delayed and accelerated in the transition to flowering. ARR was also associated with a change in PR-1 gene expression, such that young plants expressed PR-1 abundantly at 3 days post inoculation (dpi) while mature plants expressed much less. The Arabidopsis ARR response requires SA accumulation via isochorismate synthase (ICS1) [24]. ICS1 was expressed one dpi with virulent and avirulent Pst in both young and mature plants. The ARR response was also effective versus avirulent Pst providing an additional 4-fold limitation in bacterial growth. Arabidopsis ARR was found to be ineffective against two necrotrophs, Erwinia carotovora subspecies carotovora (bacterium) and Botrytis cinerea (fungus) and one obligate biotroph, Erysiphe cichoracearum (fungus). However, mature wild type, SA-deficient sid2 and NahG plants supported little growth of the obligate biotrophic oomycete, Peronospora parasitica. Therefore ARR to P. parasitica appears to be SA-independent, however the level of ARR resistance was somewhat reduced in these mutants in some experiments. Thus, there may be numerous defence pathways that contribute to adult plant resistance in Arabidopsis.     

Histochemical studies on the accumulation of reactive oxygen species (O2− and H2O2) in the incompatible and compatible interaction of wheat—Puccinia striiformis f. sp. tritici

The generation and accumulation of reactive oxygen species (ROS), superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂), were studied in the interaction between wheat cv. ‘Suwon 11’ and two races of Puccinia striiformis f. sp. tritici (avirulent and virulent). Generation of O₂⁻ and H₂O₂ was analyzed histochemically using nitroblue tetrazolium (NBT) and 3,3-diamino-benzidine (DAB), respectively. At the pre-penetration stage during appressorium formation both stripe rust races induced H₂O₂ accumulation in guard cells. In the incompatible interaction, a rapid increase of O₂⁻ and H₂O₂ generation at infection sites was detected. The percentage of infection sites showing NBT and DAB staining was 36.1% and 40.0%, respectively, 12 h after inoculation (hai). At extended incubation time until 24 hai, percentage of infection sites showing H₂O₂ accumulation further increased, whereas those exhibiting O₂⁻ accumulation declined. The early infection stage from 12 to 24 hai coincided with primary haustoria formation in mesophyll cells. In contrast, in the compatible interaction, O₂⁻ and H₂O₂ generation could not be detected in most of the infection sites. In the incompatible interaction, intensive DAB staining was also determined in mesophyll cells, especially in cell walls, surrounding the infected cells 16–24 hai; thereafter, these cells contained fluorescing compounds and underwent hypersensitive response (HR). The number of necrotic host cells surrounding the infection sites increased continuously from 20 to 96 hai. It might be concluded that H₂O₂ accumulation during the early infection stage is associated with the occurrence of hypersensitive cell death and that resistance response is leading to arrest the avirulent race of the obligate stripe rust pathogen. In the compatible interaction at 96 hai, H₂O₂ accumulation was observed in mesophyll cells surrounding the rust lesion.     

Management and cross-infectivity potential of Colletotrichum acutatum causing anthracnose on bell pepper in Florida

Early anthracnose caused by Colletotrichum acutatum has become an increasingly serious disease on green, unripe bell pepper fruit in Florida. This contrasts with earlier reports of anthracnose occurring on bell pepper primarily as a ripe-rot disease of mature, colored pepper fruit caused by Colletotrichum gloeosporioides. Management of anthracnose on green bell pepper fruit using fungicides and a commercial inducer of systemic acquired resistance, acibenzolar-S-methyl (ASM), was evaluated during three seasons. In two of the three trials, all the fungicides tested including azoxystrobin, fludioxonil + cyprodinil, mancozeb, famoxadone + cymoxanil, copper hydroxide, and ASM significantly increased the number of marketable fruit compared with control plants. These trials identified fungicides that could contribute to a successful pest management program on pepper for controlling anthracnose caused by C. acutatum. The cross-infectivity potential of C. acutatum was investigated on tomato and strawberry by in vitro and field inoculation. Anthracnose lesions formed readily on wound-inoculated detached fruits of all hosts in in vitro assays. Under field conditions, after inoculation, anthracnose lesions occurred on pepper fruit but no lesions of anthracnose were found on either ripe or unripe tomato or strawberry fruit in adjacent plots.     

Cross protection provides evidence for race-specific avirulence factors inFusarium oxysporum

Simultaneous inoculation with races 1 and 2 of the vascular wilt pathogenFusarium oxysporumf.sp.lycopersiciprovided a high level of protection against race 2 in three tomato cultivars carrying resistance geneI, which confers resistance to race 1 but not race 2. However, simultaneous inoculation did not provide any protection in cultivars lacking this gene. Protection resulted in reduction and delay of wilt symptoms. Similarly, avirulent races ofF. oxysporumf.sp.melonisprotected muskmelon plants against virulent races of the sameforma specialis.A ratio 10:1 between spore concentrations of inducer and challenger organism gave the highest cross protection, but ratio 0.1:1 still provided significant disease reduction. Cross protection was also obtained when inoculation with the inducer organism was performed 6 or 12 h before inoculation with the challenger organism. Autoclaved spores of the inducer did not have any protective effect, indicating that living propagules were required to initiate protection. The results suggest the presence of a gene-for-gene interaction betweenF. oxysporumf.sp.lycopersici-tomato andF. oxysporumf.sp.melonis-muskmelon, in which cross protection against a virulent race is mediated by recognition of a specific elicitor from the avirulent race by the plant resistance gene product and by subsequent induction of the plant defense reaction.     

The role of salicylic acid in defense response of tomato to root-knot nematodes

Salicylic acid (SA) is involved in hypersensitive reactions of plants to incompatible pathogens and in systemic acquired resistance (SAR) after the attack of necrosis-inducing pests. The possible involvement of SA in defense responses of tomato to root-knot nematodes (Meloidogyne spp., RKNs) was investigated. SA was found not to be responsible for the inhibition of catalase (CAT) detected in the early stages of Meloidogyne-tomato incompatible interactions. CAT extracted from leaves was inhibited only after treatment of the seedlings with SA concentrations as high as 4 mM. Most of the amount of free SA found in plants after SA treatment was detected in the leaves. SA (0.2 mM) was found to cause a competitive inhibition of CAT only at high substrate (H₂O₂) concentrations. Under different conditions it did not affect, or even enhanced, the enzyme activity. Therefore, it is suggested that SA-mediated CAT inhibition does not operate early in resistance against RKN in tomato, although it might have a role in the consequent lesion formation. Plant uptake of SA was detected by immersion of roots of 1-month-old seedlings in aqueous solutions of SA and SA plus a soil humic acid. Considering the low level of free SA retained by roots, the capacity of exogenously provided SA to act as an elicitor of resistance to root pests is considered unlikely.     

Histochemical studies on the accumulation of reactive oxygen species (O2 and H2O2) in the incompatible and compatible interaction of wheat—Puccinia striiformis f. sp. tritici

The generation and accumulation of reactive oxygen species (ROS), superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂), were studied in the interaction between wheat cv. ‘Suwon 11’ and two races of Puccinia striiformis f. sp. tritici (avirulent and virulent). Generation of O₂⁻ and H₂O₂ was analyzed histochemically using nitroblue tetrazolium (NBT) and 3,3-diamino-benzidine (DAB), respectively. At the pre-penetration stage during appressorium formation both stripe rust races induced H₂O₂ accumulation in guard cells. In the incompatible interaction, a rapid increase of O₂⁻ and H₂O₂ generation at infection sites was detected. The percentage of infection sites showing NBT and DAB staining was 36.1% and 40.0%, respectively, 12 h after inoculation (hai). At extended incubation time until 24 hai, percentage of infection sites showing H₂O₂ accumulation further increased, whereas those exhibiting O₂⁻ accumulation declined. The early infection stage from 12 to 24 hai coincided with primary haustoria formation in mesophyll cells. In contrast, in the compatible interaction, O₂⁻ and H₂O₂ generation could not be detected in most of the infection sites. In the incompatible interaction, intensive DAB staining was also determined in mesophyll cells, especially in cell walls, surrounding the infected cells 16–24 hai; thereafter, these cells contained fluorescing compounds and underwent hypersensitive response (HR). The number of necrotic host cells surrounding the infection sites increased continuously from 20 to 96 hai. It might be concluded that H₂O₂ accumulation during the early infection stage is associated with the occurrence of hypersensitive cell death and that resistance response is leading to arrest the avirulent race of the obligate stripe rust pathogen. In the compatible interaction at 96 hai, H₂O₂ accumulation was observed in mesophyll cells surrounding the rust lesion.     

Effects of preinoculation with an avirulent isolate of Pyricularia grisea on infection and development of leaf blast lesions caused by virulent isolates on near-isogenic lines of Sasanishiki rice

Eight near-isogenic Sasanishiki rice lines with different genes for complete resistance to rice blast were inoculated with an avirulent isolate 72 h before inoculation with a virulent isolate of Pyricularia grisea to clarify the mechanisms of induced resistance in the leaf blades. Subsequent lesions on the leaf blades were classified as brown spots (b type), as observed on Sasanishiki BL no. 8 with resistance gene Pii, or no symptoms (HR type), as observed on Sasanishiki BL no. 4 with the gene Piz-t and on the six other lines. Lesion expansion was significantly reduced in Sasanishiki BL no. 8 compared with that in Sasanishiki BL no. 4 when the leaf blades were preinoculated with a high concentration of a conidial suspension of the avirulent isolate. Moreover, after preinoculation with the conidial suspension of the avirulent isolate in silicon rings on the leaf blades, induced resistance was expressed only in areas close to the inoculation sites. These resistant areas were larger in Sasanishiki BL no. 8 than in BL no. 4. Hyphal growth was markedly inhibited in the epidermal cells of Sasanishiki BL no. 8, whereas inhibition was weak in those of Sasanishiki BL no. 4. In the epidermal cells of leaf blades of Sasanishiki BL no. 8 preinoculated with the avirulent isolate, the frequency of hyphal penetration of the virulent isolate in the presence of host cell browning decreased, as did the frequency of invading hyphae after inoculation with virulent isolates. The results indicate that induced resistance may play a role in the suppression of lesion development in the Sasanishiki near-isogenic lines and that the lines differ in the extent of suppression.     

Pine Wilt Disease Caused by the Pine Wood Nematode: The Induced Resistance of Pine Trees by the Avirulent Isolates of Nematode

Since the beginning of the 20th century, pine trees in Japan have been seriously damaged by the pine wilt disease. This disease is caused by the pine wood nematode, Bursaphelenchus xylophilus, which is transmitted by the Japanese pine sawyer, Monochamus alternatus. The control of disease depends to a large extent on chemicals, but the public is now demanding environmentally friendly control methods. The virulence of B. xylophilus varies very widely. Pre-inoculation of young pine trees in a nursery with avirulent B. xylophilus has induced systemic resistance of trees against a subsequent inoculation with virulent B. xylophilus. This induced resistance was considered a hopeful means for developing a biological control for the disease. The induced resistance by the avirulent nematodes was also expressed in mature pine trees in a forest where the disease was naturally epidemic. However, the effects of induced resistance were not satisfactory for practical biological control. Since the inoculation with higher concentrations of the avirulent B. xylophilus induced the resistance more effectively, the pre-inoculation method will need to be improved to develop the biological control. The induced resistance of pine trees by avirulent B. xylophilus should be one of the candidate biological control methods against pine wilt disease. This induced resistance also provides an experimental system to clarify physiological interactions between the nematodes and pine trees.     

Comparison of six inoculation techniques withColletotrichum acutatum on cold stored strawberry plants and screening for resistance to this fungus in French strawberry collections

Six inoculation techniques were compared for their ability to evaluate resistance toColletotrichum acutatum of five strawberry cultivars. Inoculation by dipping the whole cold stored plants in a suspension of conidia adjusted to 2.10⁶ conidia ml^–1 made it possible to screen cultivars resistant to crown rot at 28 days after inoculation. Using the dipping technique, 44 strawberry cultivars were evaluated for their resistance to one strain ofC. acutatum, 1267b. Twelve of them did not show wilt symptoms and could be classified as resistant. When another strain ofC. acutatum, 494a, was inoculated to seven cultivars, all of them including Dover, resistant to 1267b, showed wilt symptoms. This result showed the importance of investigations on genotype × isolate interactions to conduct an efficient breeding programme for screening resistance toC. acutatum.     

Chlorocholine chloride mediated resistance mechanism and protection against leaf spot disease of Stevia rebaudiana Bertoni

Investigations have been carried out to determine the influence of chlorocholine chloride on induction of the resistance mechanisms of Stevia rebaudiana against leaf spot disease, caused by Alternaria alternata. The paper also focuses an impact of chlorocholine chloride induced resistance on reduction of leaf spot disease. Chlorocholine chloride is attributed to its significant role in defence responses through augmentation of phenol and salicylic acid content as well as stimulation of phenylalanine ammonia lyase and peroxidase activity in S. rebaudiana following inoculation with A. alternata. Histochemical studies revealed that fungal invasion as well as infection process was appreciably reduced in chlorocholine chloride treated plants through peroxidase-H₂O₂ mediated strengthening of cell wall. The overall study highlights the significant role of chlorocholine chloride in induction of resistance in S. rebaudiana against A. alternata.     

Induction of systemic resistance by a hypovirulent Rhizoctonia solani isolate in tomato

A polynucleate Rhizoctonia isolate (R3) was analysed for virulence, growth characteristics, enzyme production and presence of dsRNAs. Taxonomic position was assessed morphologically and by anastomosis group (AG) testing and ITS sequence analysis. Results indicated that R3 is a hypovirulent R. solani AG 4. Mechanisms underlying biocontrol towards virulent R. solani and Botrytis cinerea were investigated and plant-mediated resistance was followed using biochemical markers of defence (PR1, laminarinase, chitinase). Control apparently relies on spatial and nutrient competition in soil, and on systemic induced resistance. This is the first report on induction of systemic resistance and of defence markers by a hypovirulent strain of R. solani.     

Diversity in Puccinia triticina detected on wheat from 2008 to 2010 and the impact of new races on South African wheat germplasm

Samples of wheat and triticale infected with leaf rust were collected from 2008 to 2010 in South Africa to identify Puccinia triticina races. Races were identified based on their virulence profile on standard differential lines. Eight races were identified from 362 isolates. The dominant races were 3SA133 (syn. PDRS) in 2008 (78 %) and 2009 (34 %), and 3SA145 (47 %) in 2010. Race 3SA145 (CCPS) identified in 2009 was a new race in South Africa with virulence for the adult plant resistance gene Lr37. Another new race, 3SA146 (MCDS), was identified in 2010. Race 3SA146 is also virulent for Lr37 but unlike 3SA145, it is virulent for Lr1 and Lr23 and avirulent for Lr3ka and Lr30. Microsatellite analysis showed that 3SA145 and 3SA146 shared 70 % genetic similarity with each other, but only 30 % similarity with other races in South Africa, suggesting that both represent foreign introductions. In seedling tests of 98 South African winter and spring cultivars and advanced breeding lines, 27 % were susceptible to 3SA145 and 3SA146 but resistant to 3SA133. In greenhouse studies of 59 spring wheat adult plants, 19 % of breeding lines and 46 % of cultivars were susceptible to 3SA145, whereas 29 % of the lines and 53 % of cultivars were susceptible to 3SA146. The cssfr6 gene-specific DNA marker confirmed the presence of Lr34 gene for leaf rust resistance in a homozygous condition in 28 wheat entries. Five entries were heterogeneous for Lr34. Several entries which were susceptible as seedlings to the new races carried Lr34. These lines are expected to show lower levels of leaf rust as adult plants. Results of these studies indicate a continued vulnerability of South African wheat cultivars to new races and emphasise the importance of regular rust monitoring and the need to incorporate genes for durable resistance.     

Physiological response of Bacillus cereus C1L-induced systemic resistance in lily against Botrytis leaf blight

Bacillus cereus C1L has been demonstrated to induce systemic disease resistance against Botrytis elliptica in lily. The objective of this study was to investigate physiological responses of B. cereus C1L-triggered systemic resistance in lily cv. Star Gazer against B. elliptica. By histological and biochemical analyses, leaves inoculated with B. elliptica displayed cell death, H₂O₂ accumulation and lignin deposition. As plants were elicited with B. cereus C1L, cell death, H₂O₂ accumulation and lignin deposition in leaves caused by B. elliptica infection were suppressed, revealing that suppression of oxidative burst might be associated with B. cereus C1L-induced systemic resistance. In reactive oxygen species inhibitors assays, B. elliptica-caused lesion numbers and H₂O₂ accumulation in lily leaves were significantly reduced as leaves were pretreated with catalase or diphenylene iodonium. Furthermore, the expression of LsGRP1 and LsPsbR in leaves elicited with B. cereus C1L and inoculated with B. elliptica was decreased. The same expression pattern was also observed in leaves pretreated with catalase or diphenylene iodonium and inoculated with B. elliptica. These results suggest that B. cereus C1L-induced systemic resistance may be related to suppression or alleviation of oxidative stress and cell death of lily caused by B. elliptica.     

Induction of systemic disease resistance and pathogen defence responses in Asparagus officinalis inoculated with nonpathogenic strains of Fusarium oxysporum

The ability of nonpathogenic isolates of Fusarium oxysporum (np Fo ) to induce systemic resistance and defence responses against subsequent challenge with a pathogenic strain of F. oxysporum f. sp. asparagi ( Foa ) was examined in Asparagus officinalis . In a split-root experiment, roots inoculated with np Fo exhibited a hypersensitive response and those subsequently inoculated with Foa displayed resistance. Induction of systemic resistance in np Fo -treated plants led to significantly fewer necrotic lesions ( P  = 0·05) and reduced Foa disease severity compared with plants not treated with np Fo . In hyphal-sandwich root inoculation experiments, activities of peroxidase and phenylalanine ammonia-lyase and lignin content were higher in np Fo -treated plants and increased more rapidly than in np Fo -untreated plants after Foa inoculation. Antifungal activity (inhibition of fungal spore germination and germ-tube growth) from exudates of roots inoculated with Foa were observed for np Fo -treated plants but not for np Fo -untreated plants. Thus, isolates of np Fo may function as inducers of systemic acquired resistance (SAR) and defence responses against Foa invasion in A. officinalis .