Studies on leaf spot disease of <Emphasis Type="Italic">Withania somnifera</Emphasis> and its impact on secondary metabolites

During an investigation of the disease profile of Withania somnifera, it was observed that leaf spot is the most prevalent disease. Repeated isolations from infected leaf tissues and pathogenicity tests showed the association of fungal pathogen identified as Alternaria alternata (Fr.) Keissler. Scanning electron microscopy showed various histological changes in the leaf tissues of infected plants. A decrease in total content of reducing sugars (20%) and chlorophyll (26.5%) was observed in diseased leaves whereas an increase was noticed in proline (25%), free amino acids (3%) and proteins (74.3%). High performance thin layer chromatography (HPTLC) analysis of secondary metabolites viz. withanolides, withaferin-A and total alkaloids of the diseased leaves vis-à-vis control revealed reduction in withaferin-A and withanolides contents by 15.4% and 76.3% respectively, in contrast to an increase in total alkaloids by 49.3%, information hitherto unreported in W. somnifera.     

Post‐infectional dynamics of leaf spot disease in Withania somnifera

Withania somnifera is a high value medicinal plant, native to Indian sub‐continent, and is extensively used in traditional systems of medicine. It is highly prone to leaf spot disease caused by Alternaria alternata, that may affect the quality of crude herbal drugs. In the present study, the accumulation of ROS (reactive oxygen species) was detected histochemically within the infected host tissue, using H₂DCFDA (2′,7′‐difluorodihydrofluorescein diacetate) and DAB (3,3′‐diaminobenzidine) staining methods. This ROS accumulation was linked with the process of disease initiation. The microscopy studies were conducted to explore the site of pathogen invasion into the host tissue. Some changes related to tissue degeneration during the infection process have also been reported. The observed changes in the form of reducing sugar and phenolic content showed an enhancement during infestation. Carbon, nitrogen and sulphur exhibited a decreasing trend with the progression of disease. Both the net rate of photosynthesis (A) and stomatal conductance (g_S) showed a similar trend except that g_S increased at the early stage of infection when the protruding fungal hyphae obstructed the stomatal closure. Further, the disintegration of chloroplasts was also observed in the infected leaves. Overall, the present study highlights a few potential targets for enhancing the disease resistance against the leaf spot pathogen of W. somnifera.     

Morphology,phylogeny and pathogenicity of Alternaria species,involved in leaf spot disease of sunflower in northern Iran

Leaf spot disease of sunflower is one of the most important foliar diseases on this crop worldwide. Several fungal groups are known to cause leaf spot disease on sunflower. Species of the genus Alternaria are the most common and serious leaf spot causing fungi on this crop. Leaf spot disease is the most destructive foliar diseases on sunflower in northern Iran; however, the identity of the causal agent remains unknown. The present study was aimed to characterise the identity of the causal agent of the disease by means of morphological and molecular data as well as to evaluate the pathogenicity of the responsible species. For this purpose, a total number of 97 fungal isolates were recovered from sunflower leaves with leaf spot disease symptoms from the sunflower fields in northwestern zone of Iran. All of the isolates were identified as Alternaria alternata based on cultural and morphological characteristics. A subset of isolates was subjected to phylogenetic analysis using sequence data from ITS-rDNA region, gpd and rpb2 genes. Sequence data from ITS-rDNA and gpd did not discriminate A. alternata from the other small-spored Alternaria species. A phylogeny inferred using sequence data from rpb2 gene clustered our isolates in several sub-clades within a single monophyletic clade. Sequence data for the type strain of the other small-spored Alternaria species has to be included in phylogenetic analysis, in order to make sure, whether the observed variations in rpb2 gene sequences are an indication for the population variation in sunflower isolates of A. alternata or define species boundaries among the small-spored Alternaria species. The results of pathogenicity assay on sunflower plants (cultivar Euroflor) under greenhouse condition revealed that A. alternata is pathogenic on sunflower.     

Fungal foliar diseases in Withania somnifera and its effect on secondary metabolites

Withania somnifera is a promising revitalizing medicinal herb. The plant is affected by foliar diseases in Lakkavalli forest region of Bhadra Wildlife Sanctuary. The symptomatology of foliar fungal disease incidence, severity and distribution in the study area was examined during 2006–2009. The seedborne nature and transmission of the causal pathogen and its management with seed dressing fungicides were studied. The results of the study indicated that Alternaria alternata caused severe leaf spot disease, while Myrothecium roridum and Fusarium oxysporum caused minor diseases. Based on the internal transcribed spacer (ITS1 and ITS2) regions of rDNA, the major pathogen was identified as A. alternata. The disease is homogeneously distributed in Lakkavalli forest region and high severity is recorded during November. Alternaria alternata and Fusarium oxysporum were the dominant seedborne pathogens that are transmitted to seedlings. Among the seed dressing fungicides used, Hyzeb was the most effective, followed by Captra, Antracol and Bavistin, in reducing the incidence of A. alternata and other seedborne fungi. The infected W. somnifera foliages had decreased steroids and alkaloids and increased phenolics and flavonoids. Analysis of alkaloids in diseased foliages by high performance thin layer chromatography indicated the occurrence of transformed compounds at R_f = 0.1, 0.77 (254 nm) and 0.2 (366 nm).     

Phosphate excess increases susceptibility to pathogen infection in rice

Phosphorus (P) is an essential nutrient for plant growth and productivity. Due to soil fixation, however, phosphorus availability in soil is rarely sufficient to sustain high crop yields. The overuse of fertilizers to circumvent the limited bioavailability of phosphate (Pi) has led to a scenario of excessive soil P in agricultural soils. Whereas adaptive responses to Pi deficiency have been deeply studied, less is known about how plants adapt to Pi excess and how Pi excess might affect disease resistance. We show that high Pi fertilization, and subsequent Pi accumulation, enhances susceptibility to infection by the fungal pathogen Magnaporthe oryzae in rice. This fungus is the causal agent of the blast disease, one of the most damaging diseases of cultivated rice worldwide. Equally, MIR399f overexpression causes an increase in Pi content in rice leaves, which results in enhanced susceptibility to M. oryzae. During pathogen infection, a weaker activation of defence-related genes occurs in rice plants over-accumulating Pi in leaves, which is in agreement with the phenotype of blast susceptibility observed in these plants. These data support that Pi, when in excess, compromises defence mechanisms in rice while demonstrating that miR399 functions as a negative regulator of rice immunity. The two signalling pathways, Pi signalling and defence signalling, must operate in a coordinated manner in controlling disease resistance. This information provides a basis to understand the molecular mechanisms involved in immunity in rice plants under high Pi fertilization, an aspect that should be considered in management of the rice blast disease.     

First report of Alternaria leaf spot caused by Alternaria alternata (Fries.) Kiessler on Sonchus oleraceus L. and Convolvulus arvensis L. in Algeria

Alternaria leaf spot caused by Alternaria alternata (Fries.) Kiessler was found on sow thistle (Sonchus oleraceus L.) and field bindweed (Convolvulus arvensis L.) in the experimental station of ENSA (Ecole Nationale Supérieure d'Agronomie) in Algiers, Algeria, during the winter of 2016. Necrotic spots in the form of concentric circles were observed on the leaves of both weeds with disease incidence of approximately 70% and disease severity ranging from 50% to 70%. Fungi were isolated from the infected leaves and identified as A. alternata, based on morphological and molecular analyses (using genetic marker internal transcribed spacer, ITS of rDNA). Pathogenicity tests confirmed that A. alternata is the causing agent of leaf spot disease of sow thistle and field bindweed in accordance with the original symptoms. To the best of our knowledge, this is the first report of sow thistle and field bindweed naturally infected by A. alternata in Algeria.     

CCR-NB-LRR proteins MdRNL2 and MdRNL6 interact physically to confer broad-spectrum fungal resistance in apple (Malus × domestica)

Apple leaf spot, a disease caused by Alternaria alternata f. sp. mali and other fungal species, leads to severe defoliation and results in tremendous losses to the apple (Malus × domestica) industry in China. We previously identified three RPW8, nucleotide-binding, and leucine-rich repeat domain CC_R-NB-LRR proteins (RNLs), named MdRNL1, MdRNL2, and MdRNL3, that contribute to Alternaria leaf spot (ALT1) resistance in apple. However, the role of NB-LRR proteins in resistance to fungal diseases in apple remains poorly understood. We therefore used MdRNL1/2/3 as baits to screen ALT1-inoculated leaves for interacting proteins and identified only MdRNL6 (another RNL) as an interactor of MdRNL2. Protein interaction assays demonstrated that MdRNL2 and MdRNL6 interact through their NB-ARC domains. Transient expression assays in apple indicated that complexes containing both MdRNL2 and MdRNL6 are necessary for resistance to Alternaria leaf spot. Intriguingly, the same complexes were also required to confer resistance to Glomerella leaf spot and Marssonina leaf spot in transient expression assays. Furthermore, stable transgenic apple plants with suppressed expression of MdRNL6 showed hypersensitivity to Alternaria leaf spot, Glomerella leaf spot, and Marssonina leaf spot; these effects were similar to the effects of suppressing MdRNL2 expression in transgenic apple plantlets. The identification of these novel broad-spectrum fungal resistance genes will facilitate breeding for fungal disease resistance in apple.     

The Association Between Leaf Malondialdehyde and Lignin Content and Resistance to Spot Blotch in Wheat

Spot blotch (causative pathogen Bipolaris sorokiniana (Sacc.) Shoem) is a common disease of wheat in the Eastern Gangetic Plains region of India. The association of leaf malondialdehyde and lignin contents with the severity of spot blotch disease was studied using a correlation analysis based on a population of recombinant inbred lines bred from the cross cvs. Yangmai 6 (resistant) × Sonalika (susceptible). The material was field‐tested over two consecutive years and inoculated artificially with a highly virulent strain of the pathogen. Disease severity was assessed at three growth stages around and after anthesis. Leaf lignin content tended to be higher in the more resistant RILs, while the opposite was the case for leaf malondialdehyde content. Lesion size showed a positive correlation with disease severity and leaf malondialdehyde content, while disease severity and leaf lignin content were negatively correlated with one another, as were leaf malondialdehyde and leaf lignin content. Leaf malondialdehyde and/or leaf lignin content could be informative as markers for selection for higher levels of resistance against spot blotch in wheat.     

Antioxidant activities of red versus green leaves in Elatostema rugosum

Anthocyanin biosynthesis in leaves increases under stresses which also generate reactive oxygen species (ROS). In the present study the hypothesis that red leaves are better equipped to scavenge ROS than green leaves was tested. Antioxidants in leaf extracts from red and green morphs of Elatostema rugosum were identified, and activities quantified using enzymatic and α,α‐diphenyl‐β‐picrylhydrazyl (DPPH) assays and cyclic voltammetry. Red leaves from E. rugosum held greater amounts of superoxide dismutase, catalase, anthocyanins, and hydroxycinnamic acids, were significantly more effective at scavenging DPPH radicals, and produced higher voltammetric currents than green leaves. Anthocyanins contributed to the antioxidant pool more than all other constituent phenolics. Anthocyanin concentrations, and antioxidant activities declined with leaf age. Purified anthocyanin fractions displayed oxidative activities at both pH 7·0 and pH 5·5. Implications of the antioxidant potential of anthocyanin in its cytoplasmic and vacuolar locations are discussed.     

First Report of Black Leaf Spot Caused by Alternaria alternata on Ramie in China

In 2012 and 2013, black leaf spot disease was observed on ramie plants in Hunan and Hubei Provinces, China. In the field, the symptoms of this disease included dark green to black big spots on leaves, often resulting in upwardly curled leaf margins. The pathogen isolates were identified as Alternaria alternata (Fr.) Keissler on the basis of morphology and sequence similarity of 99–100% to the published data for internal transcribed spacer (ITS), and glyceraldehyde‐3‐phosphate dehydrogenase (gdp). To our knowledge, this is the first report of Alternaria leaf spot of ramie in China.     

CCR1, an enzyme required for lignin biosynthesis in Arabidopsis,mediates cell proliferation exit for leaf development

After initiation, leaves first undergo rapid cell proliferation. During subsequent development, leaf cells gradually exit the proliferation phase and enter the expansion stage, following a basipetally ordered pattern starting at the leaf tip. The molecular mechanism directing this pattern of leaf development is as yet poorly understood. By genetic screening and characterization of Arabidopsis mutants defective in exit from cell proliferation, we show that the product of the CINNAMOYL CoA REDUCTASE (CCR1) gene, which is required for lignin biosynthesis, participates in the process of cell proliferation exit in leaves. CCR1 is expressed basipetally in the leaf, and ccr1 mutants exhibited multiple abnormalities, including increased cell proliferation. The ccr1 phenotypes are not due to the reduced lignin content, but instead are due to the dramatically increased level of ferulic acid (FeA), an intermediate in lignin biosynthesis. FeA is known to have antioxidant activity, and the levels of reactive oxygen species (ROS) in ccr1 were markedly reduced. We also characterized another double mutant in CAFFEIC ACID O‐METHYLTRANSFERASE (comt) and CAFFEOYL CoA 3‐O‐METHYLTRANSFERASE (ccoaomt), in which the FeA level was dramatically reduced. Cell proliferation in comt ccoaomt leaves was decreased, accompanied by elevated ROS levels, and the mutant phenotypes were partially rescued by treatment with FeA or another antioxidant (N‐acetyl‐l ‐cysteine). Taken together, our results suggest that CCR1, FeA and ROS coordinate cell proliferation exit in normal leaf development.     

Evidence of Alternaria alternata Causing Leaf Spot of Aloe vera in Iran

A leaf spot and leaf blight disease was observed on Aloe vera plants as small, circular to oval dark brown necrotic sunken spots on leaves. Infected tissues collected from different sites in diseased fields were cultured on potato carrot agar medium, and the pathogen was identified as Alternaria alternata on the basis of morphological and cultural characteristics. The conidiophores were branched, straight, golden brown, smooth‐walled, measuring up to μm long by 3 μm wide with one conidial scar. The conidia were golden brown in colour and produced in long branched chains, obclavate in shape and in short conical flask. Pathogenicity tests conducted on healthy potted aloe plants in a glasshouse showed typical leaf spot symptoms after 4–7 days. The optimal temperature for the growth of A. alternata was 25°C.     

A cAMP‐Dependent Protein Kinase Gene,aapk1, Is Required for Mycelia Growth,Toxicity and Pathogenicity of Alternaria alternata on Tobacco

The fungus Alternaria alternata is a common spot‐producing plant pathogen. During the past decade, tobacco brown spot disease caused by this fungus has became prevalent in China and lead to significant losses. To better understand the molecular pathogenesis of this fungus, the aapk1 gene encoding a cAMP‐dependent protein kinase catalytic subunit was cloned, sequenced and characterized. The aapk1 deletion mutants were identified from hygromycin‐resistant transformants by PCR strategy and confirmed by Southern blot analysis and RT‐PCR. The aapk1 deletion mutant exhibited reduced vegetative growth and was less toxic than the wild‐type strain sd1. Deletion of aapk1 also delayed disease development on detached tobacco leaves. Thus, we propose that the cAMP signalling pathway is involved in mycelia growth and pathogenic phenotype of Alternaria alternata.     

Alternaria alternata f.sp. cucurbitae, the cause of a new leaf spot disease of melon (Cucumis melo)

A leaf spot disease of melon caused by Alternaria alternata f.sp. cucurbitae was recorded for the first time in Crete. Necrotic flecks surrounded by chlorotic halos developed on the cotyledons and the leaves of the middle and the upper part of the plants; the flecks enlarged and coalesced to form lesions of 2 cm or more in diameter with brown fructifications of the pathogen on their surface. Severely affected cotyledons and leaves became chlorotic and died. Of 16 species from eight botanical families that were inoculated, only those of the Cucurbitaceae were susceptible. Of four isolates of A. alternata from tomato, sunflower, pear and cucumber, only the cucumber isolate was pathogenic to melon foliage.     

Volatile-mediated signalling in barley induces metabolic reprogramming and resistance against the biotrophic fungus Blumeria hordei

• Plants have evolved diverse secondary metabolites to counteract biotic stress. Volatile organic compounds (VOCs) are released upon herbivore attack or pathogen infection. Recent studies suggest that VOCs can act as signalling molecules in plant defence and induce resistance in distant organs and neighbouring plants. However, knowledge is lacking on the function of VOCs in biotrophic fungal infection on cereal plants.
• We analysed VOCs emitted by 13 ± 1-day-old barley plants (Hordeum vulgare L.) after mechanical wounding using passive absorbers and TD-GC/MS. We investigated the effect of pure VOC and complex VOC mixtures released from wounded plants on the barley–powdery mildew interaction by pre-exposure in a dynamic headspace connected to a powdery mildew susceptibility assay. Untargeted metabolomics and lipidomics were applied to investigate metabolic changes in sender and receiver barley plants.
• Green leaf volatiles (GLVs) dominated the volatile profile of wounded barley plants, with (Z)-3-hexenyl acetate (Z3HAC) as the most abundant compound. Barley volatiles emitted after mechanical wounding enhanced resistance in receiver plants towards fungal infection. We found volatile-mediated modifications of the plant–pathogen interaction in a concentration-dependent manner. Pre-exposure with physiologically relevant concentrations of Z3HAC resulted in induced resistance, suggesting that this GLV is a key player in barley anti-pathogen defence.
• The complex VOC mixture released from wounded barley and Z3HAC induced e.g. accumulation of chlorophyll, linolenic acid and linolenate-conjugated lipids, as well as defence-related secondary metabolites, such as hordatines in receiving plants. Barley VOCs hence induce a complex physiological response and disease resistance in receiver plants.

     

Reducing AsA Leads to Leaf Lesion and Defence Response in Knock-Down of the AsA Biosynthetic Enzyme GDP-D-Mannose Pyrophosphorylase Gene in Tomato Plant

As a vital antioxidant, L-ascorbic acid (AsA) affects diverse biological processes in higher plants. Lack of AsA in cell impairs plant development. In the present study, we manipulated a gene of GDP-mannose pyrophosphorylase which catalyzes the conversion of D-mannose-1-P to GDP-D-mannose in AsA biosynthetic pathway and found out the phenotype alteration of tomato. In the tomato genome, there are four members of GMP gene family and they constitutively expressed in various tissues in distinct expression patterns. As expected, over-expression of SlGMP3 increased total AsA contents and enhanced the tolerance to oxidative stress in tomato. On the contrary, knock-down of SlGMP3 significantly decreased AsA contents below the threshold level and altered the phenotype of tomato plants with lesions and further senescence. Further analysis indicated the causes for this symptom could result from failing to instantly deplete the reactive oxygen species (ROS) as decline of free radical scavenging activity. More ROS accumulated in the leaves and then triggered expressions of defence-related genes and mimic symptom occurred on the leaves similar to hypersensitive responses against pathogens. Consequently, the photosynthesis of leaves was dramatically fallen. These results suggested the vital roles of AsA as an antioxidant in leaf function and defence response of tomato.