Arbuscular mycorrhiza‐mediated resistance in tomato against Cladosporium fulvum‐induced mould disease

Root colonization with arbuscular mycorrhizal fungi (AMF) enhances plant resistance particularly against soil‐borne pathogenic fungi. In this study, mycorrhizal inoculation with Glomus mosseae (Gm) significantly alleviated tomato mould disease caused by the air‐borne fungal pathogen, Cladosporium fulvum (Cf). The disease index (DI) in local leaves (receiving pathogen inoculation) and systemic leaves (just above the local leaf without pathogen inoculation) was 36.4% and 11.7% in mycorrhizal plants, respectively, whereas DI was 59.6% and 36.4% in the corresponding leaves of AMF non‐inoculated plants, after 50 days of Gm inoculation, corresponding to 15 days after Cf inoculation by leaf infiltration. Foliar spray inoculation with Cf also revealed that AMF pre‐inoculated plants had a higher resistance against subsequent pathogen infection, where the DI was 41.3% in mycorrhizal plants vs. 64.4% in AMF non‐inoculated plants. AMF‐inoculated plants showed significantly higher fresh and dry weight than non‐inoculated plants under both control (without pathogen) and pathogen treatments. AMF‐inoculated plants exhibited significant increases in activities of superoxide dismutase and peroxidase, along with decreases in levels of H₂O₂ and malondialdehyde, compared with non‐inoculated plants after pathogen inoculation. AMF inoculation led to increases in total chlorophyll contents and net photosynthesis rate as compared with non‐inoculated plants under control and pathogen infection. Pathogen infection on AMF non‐inoculated plants led to decreases in chlorophyll fluorescence parameters. However, pathogen infection did not affect these parameters in mycorrhizal plants. Taken together, these results indicate that AMF colonization may play an important role in plant resistance against air‐borne pathogen infection by maintaining redox poise and photosynthetic activity.     

Age-related Resistance in Bell Pepper to Cucumber mosaic virus

We demonstrated the occurrence of mature plant resistance in Capsicum annuum‘Early Calwonder’ to Cucumber mosaic virus (CMV) under greenhouse conditions. When Early Calwonder plants were sown at 10 day intervals and transplanted to 10‐cm square pots, three distinct plant sizes were identified that were designated small, medium and large. Trials conducted during each season showed that CMV accumulated in inoculated leaves of all plants of each size category. All small plants (with the exception of the winter trial) developed a systemic infection that included accumulation of CMV in uninoculated leaves and severe systemic symptoms. Medium plants had a range of responses that included no systemic infection to detection of CMV in uninoculated leaves with the systemically infected plants being either symptomless or expressing only mild symptoms. None of the large plants contained detectable amounts of CMV in uninoculated leaves or developed symptoms. When plants were challenged by inoculation of leaves positioned at different locations along the stem or different numbers of leaves were inoculated, large plants continued to accumulate CMV in inoculated leaves but no systemic infection was observed. When systemic infection of large plants did occur, e.g. when CMV‐infected pepper was used as a source of inoculum, virus accumulation in uninoculated leaves was relatively low and plants remained symptomless. A time‐course study of CMV accumulation in inoculated leaves revealed no difference between small and large plants. Analyses to examine movement of CMV into the petiole of inoculated leaves and throughout the stem showed a range in the extent of infection. While all large plants contained CMV in inoculated leaves, some had no detectable amounts of virus beyond the leaf blade, whereas others contained virus throughout the length of the stem but with limited accumulation relative to controls.     

Changes in specific activities of peroxidase, chitinase and phenylalanine ammonia-lyase and phenolic content in cucumber leaves inoculated with Podosphaera fusca, the causal agent of powdery mildew

In this investigation, the effects of powdery mildew disease [caused by Podosphaera fusca (syn. Sphaerotheca fuliginea)] on specific activities of several defense-related enzymes and phenolic content were studied in cucumber leaves. Spore suspension of the fungus was sprayed on cucumber (cv. Super Dominus) plants in greenhouse and leaves from both inoculated and non-inoculated control plants were sampled at 0, 24, 48, 72 and 144 hours after inoculation (HAI). Spore germination and tissue colonization of P. fusca were microscopically studied on the inoculated surface of leaf samples. Further, Phenolic content (PHE) and specific activities of peroxidase (POX), chitinase (CHI) and phenylalanine ammonia-lyase (PAL) were spectrophotometrically measured in leaf extracts. Time-course of disease progress on the leaf surface showed that maximum spore germination occurred within 24 HAI and host penetration and disease development process began during 24-48 HAI. Evaluation of enzyme activities showed that POX specific activity in inoculated plants significantly increased at 72 HAI onwards and reached 2.5 times of that of control at 144 HAI. CHI specific activity showed a transient reduction in inoculated plants between 48-72 HAI and thereafter increased significantly in relation to control. PAL specific activity in inoculated plants was not significantly different from that of control. PHE in inoculated plants showed a significant increase compared to control at 48 HAI and thereafter. Comparison of time-course of disease progress with changes in enzyme activities indicated that POX activity had an increasing trend during disease progress whereas CHI activity showed a transient decrease at the early stages and then increased during the later stages of infection: PAL activity did not show any changes during the infection and PHE increased at the early stages of infection process and remained constant at rest of the time.     

Silicon-Induced changes in antifungal phenolic acids, flavonoids, and key phenylpropanoid pathway genes during the interaction between miniature roses and the biotrophic pathogen Podosphaera pannosa

Application of 3.6 mm silicon (Si+) to the rose (Rosa hybrida) cultivar Smart increased the concentration of antimicrobial phenolic acids and flavonoids in response to infection by rose powdery mildew (Podosphaera pannosa). Simultaneously, the expression of genes coding for key enzymes in the phenylpropanoid pathway (phenylalanine ammonia lyase, cinnamyl alcohol dehydrogenase, and chalcone synthase) was up-regulated. The increase in phenolic compounds correlated with a 46% reduction in disease severity compared with inoculated leaves without Si application (Si-). Furthermore, Si application without pathogen inoculation induced gene expression and primed the accumulation of several phenolics compared with the uninoculated Si- control. Chlorogenic acid was the phenolic acid detected in the highest concentration, with an increase of more than 80% in Si+ inoculated compared with Si- uninoculated plants. Among the quantified flavonoids, rutin and quercitrin were detected in the highest concentrations, and the rutin concentration increased more than 20-fold in Si+ inoculated compared with Si- uninoculated plants. Both rutin and chlorogenic acid had antimicrobial effects on P. pannosa, evidenced by reduced conidial germination and appressorium formation of the pathogen, both after spray application and infiltration into leaves. The application of rutin and chlorogenic acid reduced powdery mildew severity by 40% to 50%, and observation of an effect after leaf infiltration indicated that these two phenolics can be transported to the epidermal surface. In conclusion, we provide evidence that Si plays an active role in disease reduction in rose by inducing the production of antifungal phenolic metabolites as a response to powdery mildew infection.     

Infection of Blackcurrant Leaves by Drepanopeziza ribis in Relation to Weather Conditions and Leaf Position

Drepanopeziza ribis causes the leaf spot disease of blackcurrant ( Ribes nigrum ) and may lead to severe premature leaf-fall. Artificial inoculation studies were carried out to investigate infection of leaves by D. ribis conidia in relation to environmental conditions and leaf position (age) on cvs. Baldwin and Ben Hope in April and July 2007. All leaves on a number of selected extension shoots on potted three-year old plants were inoculated with conidia and then incubated under different conditions: 10, 17.5 and 25°C each with five wet periods (4, 8, 12, 24 and 30 h). Number of infected leaves was determined. The two cultivars differed significantly in their susceptibility to conidial infection: cv. Baldwin was much more susceptible than cv. Ben Hope. Older leaves on extension shoots were more susceptible to conidial infection than younger leaves. Increasing length of wetness duration led to increasing incidence of leaves infected, particularly when inoculated in July. However, the effects of temperature were inconclusive and generally very small in comparison with other factors. Field epidemics were monitored over three years (2005–07). Field data confirmed the main findings from controlled inoculation studies: severe disease was associated with very wet conditions and older leaves. Furthermore, they also suggested that significant disease increase only occurred from late July onwards.     

Factors affecting aphid acquisition and transmission of soybean mosaic virus

Myzus persicae transmitted soybean mosaic virus (SMV) most efficiently following 30 or 60 s acquisition probes on infected plants. There were no differences in susceptibility to SMV infection of soybean plants 1 to 12 wk old, but symptoms were more severe in plants inoculated when young than when old. Soybeans inoculated between developmental stages R3 and R6 only showed yellowish-brown blotching on one or more leaves. There were no observable differences in the time of appearance or type of symptoms shown by soybean seedlings inoculated either by sap or by aphids; infected plants became acquisition hosts for aphids 5–6 days after inoculation. There was no change in the efficiency with which M. persicae transmitted SMV from source plants up to 18 wk after inoculation. M. persicae transmitted SMV from leaves of field-grown soybeans when plants were inoculated at developmental stages V6, R2, and R3 and tested as sources 57–74 days after inoculation but not from plants inoculated at R5 and tested as sources 14 to 32 days after inoculation. M. persicae acquired SMV from soybean buds, flowers, green bean pods, and unifoliolate, trifoliolate, and senescent leaves. Middle-aged and deformed leaves were better sources of the virus than buds, unfolding and old symptomless leaves. The results are being incorporated into a computer model of SMV epidemiology.     

STEM INFECTION OF COTTON BY XANTHOMONAS MALVACEARUM (E. F. SM.) DOWSON

Varying the position of stem inoculation, the concentration of inoculum and the age of plant affected the reaction of cotton, Gossypium sp., to infection with Xanthomonas malvacearum (E. F. Sm.) Dowson.
The extent of stem discoloration, internal and external, and the probability of disease ocurring in leaves by bacteria moving within the plant increased ( a ) the nearer the point of stem inoculation was to the apex, and ( b ) the higher the concentration of inoculum. The leaf symptoms were not the angular spots typical of primary leaf infection. Instead, bacteria seemed to lodge in, discolour and blacken sections of leaf veins. Then tissue next to the affected veins became water-soaked and leaf sectors dependent upon these veins died and dried. These symptoms usually developed 14 to 55 days after inoculation in the expanding leaves.
The amounts of stem discoloration and the probabilities of leaf symptoms developing were less when hypocotyls of old plants were inoculated than when hypocotyls of young plants were inoculated. The probabilities of leaf symptoms developing were similar, however, when young tissues in young and old plants were inoculated.
American cotton, Gossypium hirsutum , was less affected by stem inoculation than Egyptian cotton, G. barbadense. Of the resistance factors against primary leaf infection only B_6m gave appreciable stem resistance.     

The effect of early blight disease caused by Alternaria solani on shoot growth of young tomato plants

The effect of increasing spore concentration of Alternaria solani (Early blight disease) on the shoot growth of young tomato plants was analysed. Changes in growth were related to the severity of infection which increased with increasing inoculum. Leaf production was not affected but dry weights and especially leaf expansion were decreased. The effective leaf areas of the five inoculated leaves (L1-L5 numbered from the plant base) were drastically decreased by expanding necrotic lesions and, to a lesser extent, by premature leaf fall. Healthy leaves expanding soon after inoculation (L6, L7) were markedly affected by the disease on the lower leaves and had decreased specific leaf areas (ratio of leaf area to leaf dry weight) but later formed (from L8) leaves were less affected and had greater specific leaf areas than equivalent leaves on uninoculated plants.     

Construction of EGFP-Labeling System for Visualizing the Infection Process of Xanthomonas axonopodis pv. citri in planta

Xanthomonas axonopodis pv. citri (Xac) is the causal agent of citrus bacterial canker, an economically important disease to world citrus industry. To monitor the infection process of Xac in different citrus plants, the enhanced green florescent protein (EGFP) visualizing system was constructed to visualize the propagation and localization in planta. First, the wild-type Xac was isolated from the diseased leaves of susceptible 'Bingtang' sweet orange, and then the isolated Xac was labeled with EGFP by triparental mating. After PCR identification, the growth kinetics and pathogenicity of the transformants were analyzed in comparison with the wild-type Xac. The EGFP-labeled bacteria were inoculated by spraying on the surface and infiltration in the mesophyll of 'Bingtang' sweet orange leaves. The bacterial cell multiplication and diffusion processes were observed directly under confocal laser scanning microscope at different intervals after inoculation. The results indicated that the EGFP-labeled Xac releasing clear green fluorescence light under fluorescent microscope showed the infection process and had the same pathogenicity as the wild type to citrus. Consequently, the labeled Xac demonstrated the ability as an efficient tool to monitor the pathogen infection.     

THE EFFECT OF TEMPERATURE ON INFECTION WITH STRAINS OF CUCUMBER MOSAIC VIRUS

Cucumber mosaic virus strains differed in their ability to multiply in plants at 37° C. Some strains multiplied in inoculated leaves and produced systemic symptoms in plants at this temperature; plants systemically infected with one such strain remained infected after prolonged treatment at 37° C. Other strains did not appear to multiply in inoculated leaves at 37° C. and heat treatment was successful in freeing plants from infection with these. Tests with one strain of each type showed both to be rapidly inactivated in expressed sap at 37° C.
Strains of cucumber mosaic virus forming small necrotic local lesions in leaves of french bean var. Canadian Wonder, produced many fewer lesions in plants kept after inoculation at 25° C. for 24 hr. and then at 15° C. than in plants kept continuously at the lower temperature.     

Effects of Salicylate on Systemic Invasion of Tobacco Plants by Various Viruses

Salicylate watered onto soil in which White Burley tobacco plants were grown represents a reversible stress characterized by stomatal closure, slight slackening of plant growth and low chlorophyll loss. Salicylate affected viral pathogenesis in opposite ways. It had no effect against local and systemic infections by potato virus X (PVX), potato virus Y⁰ (PVY⁰) or tobacco mosaic virus (TMV), whereas it completely prevented systemic infection by alfalfa mosaic virus (AIMV) or tobacco, rattle virus (TRV) in a high proportion of treated plants. When infection moved from leaves inoculated with AIMV or TRV, the tendency to limit systemic spread was shown by the restriction of systemic infection to very limited areas erratically distributed in some uninoculated leaves. The salicylate-induced restriction of AIMV or TRV infectivity to inoculated leaves did not appear due to inhibition of virus multiplication because the inoculation of potentially resistant leaves of salicylate-reated plants resulted in virus antigen accumulation comparable to that of untreated controls. Salicylate may therefore inhibit some long distance virus transport function. Salicylate appears able to evoke true hypersensitivity only against systemic viruses able to induce local necrotic lesions, probably by activating some genetic information for resistance that is normally not expressed.     

Subterranean clover mottle sobemovirus: its host range, resistance in subterranean clover and transmission through seed and by grazing animals

Subterranean clover mottle sobemovirus (SCMV) was transmitted by manual inoculation of sap to 27 cultivars belonging to three sub-species of subterranean clover. The virus readily infected systemically all inoculated plants of five susceptible cultivars of ssp. subterraneum. Ten others showed partial resistance as not all infected plants developed systemic infection; cold winter conditions further delayed or prevented systemic movement in four of them. Two cultivars of spp. brachycalycinum and four of spp. yanninicum failed to develop systemic infection following inoculation and were considered highly resistant. Resistance to SCMV in three of the spp. yanninicum was further confirmed by the failure to establish detectable primary infections in most of the inoculated leaves. Moreover, when the four ssp. yanninicum cultivars were graft-inoculated with SCMV, systemic infection eventually developed in them but the virus concentration was low. SCMV was also transmitted by manual inoculation of sap to a further 23 species of Trifolium, Medicago or Pisum. Three species were non-hosts, five were infected only in inoculated leaves and 18 others developed systemic infection in some or all plants. SCMV reached very high concentrations and was stable in subterranean clover sap. It was transmitted experimentally between subterranean clover plants by brushing infected leaves against healthy ones and in swards was readily transmitted by the trampling and grazing of sheep, but only poorly by mowing. Seed transmission of SCMV to seedlings of five cultivars of subterranean clover was low (0–0.12%). SCMV was not transmitted by Myzus persicae.     

Physiology of Rice Tungro Virus Disease: Increased Cytokinin Activity in Tungro-Infected Rice Cultivars

Tungro virus infection decreased the chlorophyll content in the susceptible rice (Oryza sativa) cultivar ‘Taichung Native 1’, while it increased the pigment in the less-susceptible cultivar ‘IR 20’ during the later stages of infection. The virus was recovered from the infected susceptibile cultivar as early as 3 days after inoculation. Relatively low amounts of virus were recovered from the less-susceptible cultivar between 12 and 30 days after inoculation. The senescence of detached leaves of virus-infected less-susceptible cultivar incubated in water in darkness was considerably delayed compared to healthy leaves. The cytokinin activity in the extracts from inoculated less-susceptible cultivar was 69% higher than in the extracts from the healthy plants.     

Local and systemic spread of tobacco mosaic virus in transgenic tobacco.

Expression of a chimeric gene encoding the coat protein (CP) of tobacco mosaic virus (TMV) in transgenic tobacco plants confers resistance to infection by TMV. We investigated the spread of TMV within the inoculated leaf and throughout the plant following inoculation. Plants that expressed the CP gene [CP(+)] and those that did not [CP(-)] accumulated equivalent amounts of virus in the inoculated leaves after inoculation with TMV-RNA, but the CP(+) plants showed a delay in the development of systemic symptoms and reduced virus accumulation in the upper leaves. Tissue printing experiments demonstrated that if TMV infection became systemic, spread of virus occurred in the CP(+) plants essentially as it occurred in the CP(-) plants although at a reduced rate. Through a series of grafting experiments, we showed that stem tissue with a leaf attached taken from CP(+) plants prevented the systemic spread of virus. Stem tissue without a leaf had no effect on TMV spread. All of these findings indicate that protection against systemic spread in CP(+) plants is caused by one or more mechanisms that, in correlation with the protection against initial infection upon inoculation, result in a phenotype of resistance to TMV.     

Resistance phenotypes of transgenic tobacco plants expressing different cucumber mosaic virus (CMV) coat protein genes

Tobacco plants expressing a transgene encoding the coat protein (CP) of a subgroup I strain of cucumber mosaic cucumovirus (CMV), I17F, were not resistant to strains of either subgroup I or II. In contrast, the expression of the CP of a subgroup II strain, R, conferred substantial resistance, but only towards strains of the same subgroup. When protection was observed, the levels of resistance were similar when plants were inoculated with either virions or viral RNA, but resistance was more effective when plants were inoculated with viruliferous aphids. Resistance was not dependent on inoculum strength and was expressed as a recovery phenotype not yet described for plants expressing a CMV CP gene. Recovery could be observed either early in infection (less than one week after inoculation) or later (4 to 5 weeks after inoculation). In plants showing early recovery, mild symptoms were observed on the inoculated leaves, and in some cases symptoms developed on certain lower systemically infected leaves, but the upper leaves were symptomless and virus-free. Late recovery corresponded to the absence of both symptoms and virus in the upper leaves of plants that were previously fully infected. Northern blot analyses of resistant plants suggested that a gene silencing mechanism was not involved in the resistance observed.     

The RustPuccinia abruptavar.partheniicola,a Potential Biocontrol Agent of Parthenium Weed: Environmental Requirements for Disease Progress

The rust fungusPuccinia abruptavar.partheniicola,a potential biological control agent of parthenium weed (Parthenium hysterophorus), was evaluated under controlled environmental conditions. A range of spore germination temperatures as well as dew period durations and temperatures were investigated to determine some of the environmental requirements for disease establishment and disease progress. Plants were inoculated with urediniospores and exposed to dew periods between 3 to 12 h at temperatures of 10, 15, or 20°C. For disease expression, the inoculated plants were then grown in a glasshouse at one of two temperature regimes (30/26°C or 18/13°C; day/night). Urediniospores germinated best at 12 ± 1°C, with lower germination rates at 5°C or above 20°C. No infection occurred when the plants were exposed to dew periods of ≤3 h, regardless of the incubation temperature. The disease progressed most rapidly when plants were inoculated and incubated for a dew period of at least 12 h at a temperature of 15 ± 1°C. The disease progressed most slowly following inoculation at dew periods of 6 h or less. Disease progress was more rapid when the plants were exposed to a cool-temperature regime (18/13°C) than when exposed to a warm-temperature regime (30/26°C). This suggests that good infection of parthenium weed could be obtained when the urediniospores arrive on the plants during the afternoon in the cooler months of the central Queensland autumn when relatively long dew periods are expected.