Cytology of induced systemic resistance of cucumber to Colletotrichum lagenarium

The infection of cucumber leaves by Colletotrichum lagenarium was studied using cytological methods. Its progress in untreated plants was compared with that in plants in which systemic resistance had been induced by pre-infecting the first true leaf with the same fungus. In induced plants, a reduction of fungal development was observed at the leaf surface, in the epidermis, and in the mesophyll. On the leaf surface, formation of appressoria was slightly reduced. In the epidermis, enhanced formation of papillae beneath appressoria, and possibly increased lignification of entire cells, correlated with reduced development of infection hyphae. Papillae contained callose, identified by staining with aniline-blue fluorochrome and digestion with -1,3-glucanase, as a main structural component. In the mesophyll, reduced fungal development provided evidence for the existence of an additional induced defence reaction. The results imply that preinfection elicited a systemic, multicomponent defence reaction of the host plant against the fungus.Dedicated to the memory of Professor H. Grisebach     

Induction of a new Lipoxygenase Form in Cucumber Leaves by Salicylic Acid or 2,6-Dichloroisonicotinic Acid*

Changes in lipoxygenase (LOX) protein pattern and/or activity were investigated in relation to acquired resistance of cucumber (Cucumis sativus L.) leaves against two powdery mildews, Sphaerotheca fuliginea (Schlecht) Salmon and Erysiphe cichoracearum DC et Merat. Acquired resistance was established by spraying leaves with salicylic acid (SA) or 2,6-dichloroisonicotinic acid (INA) and estimated in whole plants by infested leaf area compared to control plants. SA was more effective than INA. According to Western blots, untreated cucumber leaves contained a 97 kDa LOX form, which remained unchanged for up to 48 h after pathogen inoculation. Upon treatment with SA alone for 24 h or with INA plus pathogen, an additional 95 kDa LOX form appeared which had an isoelectric point in the alkaline range. For the induction of this form, a threshold concentration of 1 mM SA was required, higher SA concentrations did not change LOX-95 expression which remained similar between 24 h and 96 h but further increased upon mildew inoculation. Phloem exudates contained only the LOX-97 form, in intercellular washing fluid no LOX was detected. dichloroisonicotinic localization revealed LOX protein in the cytosol of the mesophyll cells without differences between the forms.     

Resistenzinduktion gegen systemische Virusinfektionen durch Kulturfiltrate von Stachybotrys chartarum (Ehrenb. ex Link) Hughes

Induced resistance in systemic host-virus combinations by culture filtrates of Stachybotrys chartarum (Ehrenb. ex Link) Hughes Culture filtrates of the fungus Stachybotrys chartarum sprayed on systemic hosts decreased the development of symptoms of different elongated and isometric viruses. The degree of induced resistance depended on the host-virus system. An interval of three or five days between application of the filtrate and virus inoculation was sufficient to induce resistance. High inoculum concentration reduced the efficiency of induced resistance in cucumber against CMV. The content of CMV in inoculated andsystemically infected, induced resistant cucumber leaves was decreased. TMV inoculated leaves of induced resistant tobacco plants contained higher, systemically infected leaves lower virus amounts as comparable untreated control leaves. Reduced virus content and distribution in induced resistant plants obviously resulted from inhibition of multiplication and spread of viruses.     

A Histological Evaluation of Induced Resistance to Phytophthora infestans (Mont.) de Bary in Potato Leaves

Systemically induced resistance against Phytophthora infestans in the potato cultivar ‘Bintje’ was studied histologically at the light microscopy level on the leaf surface, in the epidermis and in the mesophyll of challenged potato leaves. Systemic disease resistance was induced by a local pre-infection with the same fungus. On the leaf surface of induced plants, the germination of cysts was enhanced. In the epidermis of induced plants, papilla deposition increased and penetration decreased, whereas in the mesophyll of induced plants the spread of hyphae was reduced. It is suggested that the reduction of disease severity in induced plants is the result of the combined action of several successive defence reactions.     

RNAi of the sesquiterpene cyclase gene for phytoalexin production impairs pre- and post-invasive resistance to potato blight pathogens

Potato antimicrobial sesquiterpenoid phytoalexins lubimin and rishitin have been implicated in resistance to the late blight pathogen, Phytophthora infestans and early blight pathogen, Alternaria solani. We generated transgenic potato plants in which sesquiterpene cyclase, a key enzyme for production of lubimin and rishitin, is compromised by RNAi to investigate the role of phytoalexins in potato defence. The transgenic tubers were deficient in phytoalexins and exhibited reduced post-invasive resistance to an avirulent isolate of P. infestans, resulting in successful infection of the first attacked cells without induction of cell death. However, cell death was observed in the subsequently penetrated cells. Although we failed to detect phytoalexins and antifungal activity in the extract from wild-type leaves, post-invasive resistance to avirulent P. infestans was reduced in transgenic leaves. On the other hand, A. solani frequently penetrated epidermal cells of transgenic leaves and caused severe disease symptoms presumably from a deficiency in unidentified antifungal compounds. The contribution of antimicrobial components to resistance to penetration and later colonization may vary depending on the pathogen species, suggesting that sesquiterpene cyclase-mediated compounds participate in pre-invasive resistance to necrotrophic pathogen A. solani and post-invasive resistance to hemibiotrophic pathogen P. infestans.     

Changes in leaf,stem, and root anatomy of Chrysanthemum cv. Lillian Hoek following paclobutrazol application

Plants ofChrysanthemum cv. Lillian Hoek were treated with a paclobutrazol (PBZ) soil drench and histologically examined after 3 months. PBZ application resulted in thicker leaves, reduced stem diameter, and roots with an increased diameter and an unusual segmented appearance. Increased leaf thickness was partly due to an additional layer of palisade mesophyll, although individual palisade cells were shorter, of smaller diameter, and more tightly packed. Spongy mesophyll depth was also greater and the individual cells were more rounded and the volume of intercellular space was reduced. The narrower stems had an increased development of secondary xylem, but had a marked reduction in the number of sclerenchyma bundle caps. Increased root diameter was due to an increase in the number of rows and diameter of cortical cells. In PBZ-treated plants, root cortical cell length was 50–70% less than in untreated plants, and this reduction appeared to be associated with the segmentation of the roots. PBZ inhibited secondary vascular development in the roots. This study is similar to other relevant studies in recording thicker leaves and roots with PBZ application; however, many of the underlying anatomical changes described above have not been previously reported.     

Pathogen-induced systemic resistance in Ipomoea purpurea

Infection of Ipomoea purpurea by anthracnose, the disease caused by the fungal pathogen Colletotrichum dematium, increases resistance to subsequent infections on previously uninfected leaves. Fungal isolates varied in their levels of virulence but not in the extent to which they induced resistance. Induced resistance was equally effective against all isolates tested. Plant lines varied in the baseline level of resistance expressed in newly emerging leaves. In some lines, new leaves were poorly defended but developed resistance with maturity, even in the absence of infection. In those lines, induced resistance could not prevent anthracnose damage to young leaves, and this damage reduced plant fitness by increasing juvenile mortality and decreasing juvenile growth rates. In contrast, anthracnose damage to well-defended older leaves had no effect on juvenile growth rates. In at least one line, new leaves were well-defended, regardless of disease experience. This line did not experience reduced growth from anthracnose infection of either young or mature leaves, suggesting that lines with higher baseline levels of resistance are more fit than those dependent upon induced resistance. These results suggest that induced resistance cannot substitute for baseline local resistance in this I. purpurea population.     

Bacterial infection systemically suppresses stomatal density

Many plant pathogens gain entry to their host via stomata. On sensing attack, plants close these pores to restrict pathogen entry. Here, we show that plants exhibit a second longer term stomatal response to pathogens. Following infection, the subsequent development of leaves is altered via a systemic signal. This reduces the density of stomata formed, thus providing fewer entry points for pathogens on new leaves. Arabidopsis thaliana leaves produced after infection by a bacterial pathogen that infects through the stomata (Pseudomonas syringae) developed larger epidermal pavement cells and stomata and consequently had up to 20% reductions in stomatal density. The bacterial peptide flg22 or the phytohormone salicylic acid induced similar systemic reductions in stomatal density suggesting that they might mediate this effect. In addition, flagellin receptors, salicylic acid accumulation, and the lipid transfer protein AZI1 were all required for this developmental response. Furthermore, manipulation of stomatal density affected the level of bacterial colonization, and plants with reduced stomatal density showed slower disease progression. We propose that following infection, development of new leaves is altered by a signalling pathway with some commonalities to systemic acquired resistance. This acts to reduce the potential for future infection by providing fewer stomatal openings.     

Inducible expression of bacterio-opsin in transgenic tobacco and tomato plants

The development of new strategies to enhance resistance of plants to pathogens is instrumental in preventing agricultural losses. Lesion mimic, the spontaneous formation of lesions resembling hypersensitive response lesions in the absence of a pathogen, is a dramatic phenotype occasionally induced upon expression of certain transgenes in plants. These transgenes simulate the presence of a pathogen and, therefore, activate the plant anti-pathogen defense mechanisms and induce a state of systemic resistance. Lesion mimic genes have been successfully used to enhance the resistance of a number of different plants to pathogen attack. However, constitutive expression of these genes in plants is associated with the spontaneous formation of lesions on leaves and stems, reduced growth, and lower yield. We tested the possibility of using a wound-inducible promoter to control the expression of bacterio-opsin (bO), a transgene that confers a lesion mimic phenotype in tobacco and tomato plants when constitutively expressed. We found that plants with inducible expression of bO did not develop spontaneous lesions. Nevertheless, under controlled laboratory conditions, they were found to be resistant to infection by pathogens. The activation of defense mechanisms by the bO gene was not constitutive, and occurred in response to wounding or pathogen infection. Furthermore, wounding of transgenic tobacco plants resulted in the induction of systemic resistance to pathogen attack within 48 h. Our findings provide a promising initial assessment for the use of wound-inducible promoters as a new strategy to enhance pathogen resistance in transgenic crops by means of lesion mimic genes.     

Photosynthetic Response to Water Stress in Phaseolus vulgaris

Water stressed Phaseolus vulgaris L. plants were monitored to detect the relationships between net photosynthesis, transpiration, boundary layer plus stomatal resistance, mesophyll resistance, CO₂ compensation point, ribulose, 1,5-diphosphate carboxylase activity and leaf water potential. At full expansion, the first trifoliate leaves of greenhouse grown bean plants were subjected to water stress by withholding irrigation. Gas exchange and enzyme activity of the central trifoliolate leaflets were monitored as leaf water potential decreased. Although increased stomatal resistance appeared to be the primary causal factor of reduced net photosynthesis, increased mesophyll resistance and decreased ribulose 1,5-diphosphate carboxylase activity further documented the role of non-stomatal factors.     

Cytology of induced systemic resistance of tomato to Phytophthora infestans

The infection of tomato leaves by Phytophthora infestans was followed using cytological methods. Fungal ingress and plant reactions in untreated and induced resistant plants were studied. Systemic disease resistance was induced by a local pre-infection with the same fungus. Induction retarded fungal progress at the leaf surface, epidermis and in the mesophyll. The reduced numbers of germinated cysts indicate the presence of fungitoxic substances on the leaf surface of induced plants. Frequency of fungal penetration through the outer epidermal cell wall was reduced, but only in plants exhibiting a high level of induced resistance. Autofluor-escent material, indicating the presence of lignin-like substances, accumulated rapidly beneath some of the appressoria, but this plant response was similar in induced and non-induced plants. Staining with aniline blue indicated that callose deposition was not involved in induced resistance. Thus, none of the cytologically investigated plant reactions correlated with the reduced penetration frequency observed. In the mesophyll, however, the cytological picture corresponding to a hypersensitive reaction occurred more often in induced plants. It is concluded that reduction of disease severity by induction is the result of the combined action of several successive defence reactions.Dedicated to the memory of Professor H. Grisebach     

Induced Resistance in Hypersensitive Tobacco Against Tobacco Mosaic Virus by Injection of Intercellular Fluid from Tobacco Plants with Systemic Acquired Resistance

Acquired resistance in hypersensitive tobacco plants against tobacco mosaic virus (TMV) was induced by components occurring in the intercellular fluid (IV) obtained from virus-infected plants or by plant cell wall components. Induced resistance could be transmitted through seed to the progeny. Lesion size and number were reduced significantly when the progeny was tested by TMV-inoculation. IV was extracted from the upper uninoculated leaves of four times TMV-inoculated Nicotiana tabacum cv. ‘Xanthi’ nc plants. Injection of IV from induction-inoculated plants (SAR-IV) into leaves of healthy plants followed by TMV-infection reduced lesion size significantly. A concentration of 5 × 10^?7 g SAR-IV/ml was still active. IV from healthy plants was inactive. The IV's were partly purified by gel filtration on a Sephadex G-50 column. Some fractions were active in inducing resistance as expressed in reduction of lesion size. Fractions of control-IV were inactive. It is still unknown whether the active substances in SAR-IV are in fact cell wall fragments acting as regulatory molecules in disease resistance.     

Probable mechanical transmission of a virus-like agent from rose rosette disease-infected multiflora rose to Nicotiana species

As part of efforts to identify the causal agent of the rose rosette disease (RRD) of multiflora rose (Rosa multiflora Thunb.), root tip extracts from both symptomatic and nonsymptomatic roses were used to mechanically inoculate leaves of Nicotiana glutinosa. Pale green spots were observed along the margins of the major leaf veins only on leaves inoculated with extracts prepared from symptomatic rose plants. Light microscopy revealed abnormal development of the palisade and spongy mesophyll cells in the symptomatic tissue, although no virus‐like particles (VLPs) were observed by electron microscopy. However, VLPs were observed in cells from tissue adjacent to the leaf veins and bordered by the pale green spots. Inoculation of N. benthamiana with extracts from symptomatic N. glutinosa initially did not result in visible symptoms on N. benthamiana inoculated leaves. However, approximately 4 wk post inoculation, splitting of leaf tissue across and along major leaf veins in expanding leaves occurred. In later stages of leaf expansion some leaves split in regions not associated with veins. Light microscopy of thick sections revealed separation between palisade cells and groups of small dead cells in the mesophyll tissue of expanding systemically infected leaf blades. Electron microscopy revealed crystalline arrays in the cytoplasm of mesophyll cells. No abnormal cellular changes were observed in plants inoculated with extracts prepared from nonsymptomatic rose plants.     

Infection of Subterranean Clover (Trifolium subterraneum) by Kabatiella caulivora

Kabatiella caulivora is a serious pathogen of clover ( Trifolium ) spp. Subterranean clover ( T. subterraneum ) cv. Woogenellup was inoculated with K. caulivora , to study the attachment and germination of conidia, germ-tube penetration of the plant surface, and histochemistry and ultrastructure of changes in the host associated with lesion development. The foliar architecture caused the conidia to concentrate at the base of leaflets and on the petiolules (between the leaflets and petioles). Epidermal cells immediately beneath conidia and, occasionally, also adjacent cells developed a yellow-brown discoloration 1 day post-inoculation. Penetration appeared to be directly through the cuticle, characterized by constricted hyphae at the point of entry. No appressoria were observed. In leaves, invasion was restricted to the area proximal to the petiolule and leaf mid-rib. In petioles and petiolules, the hyphae initially remained between the epidermal cells and first layer of mesophyll cells before moving intercellularly through the mesophyll tissue towards phloem tissues. The cuticle was occasionally degraded in petiole and petiolule infections, the loss of epidermal and mesophyll cell wall components was detected, and chloroplasts and starch grains were disrupted. Plants developed macroscopic symptoms 10–11 days post-inoculation with necrotic lesions occurring on leaves, petioles and petiolules. Sporulation occurred approximately 15–18 days post-inoculation when affected plants collapsed. This information may be useful for breeding programmes aimed at selecting varieties with improved resistance to the clover scorch disease.     

Shading-induced changes in the leaf mesophyll of plants of different functional types

Changes in the structural characteristics of mesophyll induced by shading were investigated in ten species of wild plants of diverse functional types. In all plant types, shading reduced leaf thickness and density by 30–50% and total surface of mesophyll, by 30–70%. The extent and mechanisms of mesophyll structural rearrangement depended on the plant functional type. In the ruderal plants, integral parameters of mesophyll, such as the surface of cells and chloroplasts and mesophyll resistance, changed threefold predominantly because of changes in the dimensions of the cells and chloroplasts. In these plants, shading reduced the volume of chloroplasts by 30%, and the chloroplast numbers per cell declined. The competitor plants showed a twofold increase in mesophyll resistance due to a decrease in the number of photosynthesizing cells per leaf area unit. Moreover, these plants maintained constant dimensions of mesophyll cells, ratios mesophyll surface/mesophyll volume and chloroplast surface/cell surface. In stress-tolerant plants, diffusion resistance of mesophyll remained the same irrespective of the growing conditions, and mesophyll rearrangement was associated with inversely proportional changes in the dimensions of the cells and cell volume per chloroplast. Noteworthy of these plants were relatively constant chloroplasts number per cell, per leaf area unit and total surface area of chloroplasts. The nature of relationship between the mesophyll diffusion resistance and structural parameters of leaf mesophyll differed in plants of diverse functional types.     

Inhibition of OsSWEET11 function in mesophyll cells improves resistance of rice to sheath blight disease

Pathogen–host interaction is a complicated process; pathogens mainly infect host plants to acquire nutrients, especially sugars. Rhizoctonia solani, the causative agent of sheath blight disease, is a major pathogen of rice. However, it is not known how this pathogen obtains sugar from rice plants. In this study, we found that the rice sugar transporter OsSWEET11 is involved in the pathogenesis of sheath blight disease. Quantitative real‐time polymerase chain reaction (qRT‐PCR) and β‐d ‐glucuronidase expression analyses showed that R. solani infection significantly enhanced OsSWEET11 expression in leaves amongst the clade III SWEET members. The analyses of transgenic plants revealed that Ossweet11 mutants were less susceptible, whereas plants overexpressing OsSWEET11 were more susceptible, to sheath blight compared with wild‐type controls, but the yield of OsSWEET11 mutants and overexpressors was reduced. SWEETs become active on oligomerization. Split‐ubiquitin yeast two‐hybrid, bimolecular fluorescence complementation and co‐immunoprecipitation assays showed that mutated OsSWEET11 interacted with normal OsSWEET11. In addition, expression of conserved residue mutated AtSWEET1 inhibited normal AtSWEET1 activity. To analyse whether inhibition of OsSWEET11 function in mesophyll cells is related to defence against this disease, mutated OsSWEET11 was expressed under the control of the Rubisco promoter, which is specific for green tissues. The resistance of transgenic plants to sheath blight disease, but not other disease, was improved, whereas yield production was not obviously affected. Overall, these results suggest that R. solani might acquire sugar from rice leaves by the activation of OsSWEET11 expression. The plants can be protected from infection by manipulation of the expression of OsSWEET11 without affecting the crop yield.     

Contingency rules for pathogen competition and antagonism in a genetically based,plant defense hierarchy

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Induced resistance of Gossypium australe against its most abundant folivore,Bucculatrix gossypii

Induced resistance of cultivated Gossypium to its exotic, agricultural pests is well studied but little is known about whether native cottons respond to damage by endemic herbivore populations. This study examined induced responses of Gossypium australe to its most abundant folivore, Bucculatrix gossypii. Prior damage did not affect the number of new mines initiated. Survival of miners on damaged, young leaves and cotyledons was reduced compared with survival on young leaves and cotyledons of undamaged plants. However, the induced resistance was not systemic; survival of miners on older, undamaged leaves of damaged seedlings was not different from survival on older leaves of undamaged controls. This localized induced resistance did not produce an overdispersed distribution of either mines or successful mines. On the contrary, the distributions tended towards clumped, although they were not statistically distinguishable from random. Although a localized induced response affected miner survival, no effects on behaviour were observed.     

The After-effect of Wind on the Photosynthesis and Transpiration of Festuca arundinacea

The after-effect of wind on photosynthesis and transpiration of Festuca arundinacea Schreb, was determined. Following a period of exposure In a controlled environment wind tunnel the wind-treated plants showed reduced rates of photosynthesis when compared with the controls under standard conditions. Evaporation from paper model tillers was measured and the boundary layer resistance was shown to be low in all but very low wind speeds. Analysis of CO₂ and H₂O diffusion pathways indicated that mesophyll resistance in wind-treated plants was higher whilst leaf surface resistance was lower than in the controls. The high mesophyll resistance in the wind-treated plants was attributed to reduced water content.     

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.