Variation for Resistance to <i>Alternaria tenuissima</i> and Potential Structural Mechanism among Different Cultivars of <i>Chrysanthemum morifolium</i>

Black spot disease, caused by the necrotrophic fungus Alternaria tenuissima (Fr.) Wiltsh (A. tenuissima), is considered a highly destructive disease of Chrysanthemum (Chrysanthemum morifolium Ramat.). A set of 17 accessions of commercial chrysanthemum cultivars were evaluated for resistance to A. tenuissima by seedling artificial inoculation. It was found that the reaction of the accessions to artificial inoculation ranged from resistant to highly susceptible. Five varieties of chrysanthemum (‘Zhongshan Taogui’, ‘Jinba’, ‘Zhongshan Jinguan’, ‘Jinling Wanhuang’ and ‘Jinling Yangguang’) were resistant; two varieties of chrysanthemum (‘Zhongshan Xinggui’ and ‘Zhongshan Jinkui’) were moderately resistant; and others were susceptible to various degrees, four varieties of chrysanthemum (‘Zhongshan Zihe’, ‘Zhongshan Jiuhong’, ‘Zaoyihong’ and ‘Jinling Jiaohuang’) were highly susceptible, especially. Some leaf morphological features of two resistant and two highly susceptible cultivars were further researched. Trichome density, length, height, gland size and stomata density were found to be associated with plant passive resistance. Resistant varieties that were identified in present study will be promising germplasm for exploitation of breeding programmes aimed at developing A. tenuissima-resistant cultivars and increasing genetic diversity.     

Resistance to Tomato leaf curl New Delhi virus in Cucurbita spp.

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus (family Geminiviridae) first reported in India and its neighbouring countries. ToLCNDV severely affects zucchini crop (Cucurbita pepo) in the main production areas of Southern Spain since 2012. This emerging begomovirus is a serious threat to this and other cucurbit crops. Breeding resistant cultivars is the most promising method for disease control, but requires the identification of sources of resistance in the Cucurbita genus. In this work, we screened for ToLCNDV resistance a large collection of Cucurbita spp. accessions, including landraces and commercial cultivars of the main cultivated species, C. pepo, Cucurbita moschata and Cucurbita maxima and wild species. The screening was performed using mechanical and whitefly inoculation. The level of resistance was assessed by scoring symptom severity and by measuring the virus content with quantitative polymerase chain reaction in selected genotypes. Diversity in the response was observed within and among species. Severe symptoms and high viral amounts were found at 30 days after mechanical and whitefly inoculation in C. pepo, in all accessions belonging to the Zucchini morphotype and to other morphotypes of both subspecies, pepo and ovifera, and even in the wild relative Cucurbita fraterna. C. maxima was also highly susceptible. This species showed characteristic symptoms of leaf decay and intense yellowing, different from those of mosaic, curling and internode shortening found in C. pepo. The only species showing resistance was C. moschata. Four accessions were symptomless or had some plants with only mild symptoms after three independent rounds of mechanical inoculation with different inoculum sources. Two of them also remained symptomless after virus inoculation with viruliferous whiteflies. ToLCNDV was detected in these asymptomatic accessions at 15 and 30 days post inoculation, but viral amounts were much lower than those found in susceptible genotypes, suggesting a high level of resistance. The symptoms in the susceptible accessions of this species were also different, with a characteristic leaf mottling, evolving to a severe mosaic. The newly identified C. moschata resistant accessions are good candidates for breeding programmes to avoid the damage caused by ToLCNDV.     

Antioxidative enzymes and isozymes analysis of taro genotypes and their implications in <Emphasis Type="Italic">Phytophthora</Emphasis> blight disease resistance

Assessment of the differential expression of antioxidative enzymes and their isozymes, was done in 30 day-old ex vitro raised plants of three highly resistant (DP-25, Jhankri and Duradim) and one highly susceptible (N-118) genotypes of taro [Colocasia esculenta (L.) Schott]. Antioxidative enzymes were assayed in the ex vitro plants, 7 days after inoculation with the spores (15,000 spores ml⁻¹ water) of Phytophthora colocasiae Raciborski to induce taro leaf blight disease. Uninoculated ex vitro plants in each genotype were used as control. The activity of superoxide dismutase (SOD) and guaiacol peroxidase (GPX) increased under induced blight condition when compared with control. Increase in antioxidative enzymes was more (67–92%) in the resistant genotypes than that (21–29%) of the susceptible genotype. The zymograms of SOD and GPX in the resistant genotypes, with pathogenic infection, showed increased activity for anodal isoform of SOD and increased expression and/or induction of either POX 1 or POX 2 isoforms of GPX. In susceptible genotype, expression of the above isoforms was faint for SOD and nearly absent for GPX under both blight free and induced blight conditions. Induction and/or increased activity of particular isoform of SOD and GPX against infection of Phytophthora colocasiae in the resistant genotypes studied led to the apparent conclusion of linkage of isozyme expression with blight resistance in taro. This might be an important criterion in breeding of taro for Phytophthora leaf blight resistance.     

不同苹果品种对褐斑病抗性的鉴定及抗性生理研究

对42个苹果栽培品种叶片褐斑病进行了田间发病状况调查与抗性鉴定,分析了叶片气孔密度和大小与抗病性之间的关系,同时研究了不同抗性品种离体叶片接种病原菌后超氧化物歧化酶（SOD）、多酚氧化酶（vPo）、过氧化物酶（POD）、苯丙氨酸解氨酶（PAL）活性和木质素含量的差异变化。依照抗性分级标准,供试材料中抗病品种有14个（其中高抗品种2个）,感病品种有28个（其中高感品种7个）;叶片气孔密度与病情指数之间存在显著正相关,相关系数r=0．683;叶片接种后,诱导了4种酶活性和木质素含量的升高,抗病和感病品种的SOD和PP0活性无显著差异,而抗病品种的POD和PAL活性以及木质素含量显著高于感病品种。苹果叶片的气孔密度、POD和PAL的活性以及木质素含量与褐斑病抗性有关。     

Transcriptomic and Proteomic Analyses of Resistant Host Responses in Arachis diogoi Challenged with Late Leaf Spot Pathogen,Phaeoisariopsis personata

Late leaf spot is a serious disease of peanut caused by the imperfect fungus, Phaeoisariopsis personata. Wild diploid species, Arachis diogoi. is reported to be highly resistant to this disease and asymptomatic. The objective of this study is to investigate the molecular responses of the wild peanut challenged with the late leaf spot pathogen using cDNA-AFLP and 2D proteomic study. A total of 233 reliable, differentially expressed genes were identified in Arachis diogoi. About one third of the TDFs exhibit no significant similarity with the known sequences in the data bases. Expressed sequence tag data showed that the characterized genes are involved in conferring resistance in the wild peanut to the pathogen challenge. Several genes for proteins involved in cell wall strengthening, hypersensitive cell death and resistance related proteins have been identified. Genes identified for other proteins appear to function in metabolism, signal transduction and defence. Nineteen TDFs based on the homology analysis of genes associated with defence, signal transduction and metabolism were further validated by quantitative real time PCR (qRT-PCR) analyses in resistant wild species in comparison with a susceptible peanut genotype in time course experiments. The proteins corresponding to six TDFs were differentially expressed at protein level also. Differentially expressed TDFs and proteins in wild peanut indicate its defence mechanism upon pathogen challenge and provide initial breakthrough of genes possibly involved in recognition events and early signalling responses to combat the pathogen through subsequent development of resistivity. This is the first attempt to elucidate the molecular basis of the response of the resistant genotype to the late leaf spot pathogen, and its defence mechanism.     

Isozymes variability among <Emphasis Type="Italic">Fusarium udum</Emphasis> resistant cultivars of pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) (Millsp)

The present study was carried out to select the different pigeonpea cultivars for resistance against wilt caused by Fusarium udum and to assess the genetic variability among the resistant and susceptible cultivars. These cultivars were screened by root dip inoculation and classified into resistant (ICP 8863 and 9145), moderately resistant (ICP 11681 and Selection-1), susceptible (ICP 7118, TRG-1 and LRG-30) and highly susceptible cultivars (ICP-2376 and LRG-41). The peroxidase activity (PEO) in both leaf and root tissues of four pigeonpea cultivars (ICP 8863, Selection-1, ICP 2376 and LRG-30) were determined at 1^st, 4^th and 7^th day after inoculation (DAI) in healthy and F. udum infected tissues. Higher PEO activity in both leaf and root was observed and at 4^th DAI in susceptible cultivars. In native-PAGE analysis of isozymes, the induction of specific leaf peroxidase band (Em=0.17) and two root peroxidase bands (Em=0.24 and 0.55) were observed in ICP 8863 after inoculation. Significant differences were observed in the leaf phosphatase and esterase banding profiles of all the cultivars. The presence of leaf phosphatase band at Em of 0.04 was observed only in ICP 8863 and 11681. The leaf esterase band (Em=0.3) was well expressed in ICP 8863 when compared to other cultivars. The significance of peroxidase in plant defense mechanism and utility of biochemical markers in breeding programmes are discussed. Part of M.Sc. (Ag) thesis of the first author and approved by the Acharya N.G. Ranga Agricultural University during March 2002.     

Screening of mulberry germplasm lines against Powdery mildew,Myrothecium leaf spot and Pseudocercospora leaf spot disease complex

Abstract

Fifty-six indigenous and 29 exotic mulberry varieties were screened against powdery mildew, Myrothecium leaf spot, Pseudocercospora leaf spot for a period of three years under field conditions. The percent disease index (PDI) was recorded during the peak season of the diseases. Out of 85 germplasm lines studied four-germplasm lines viz. Thailand lobed (0.43), M. multicaulis (2.63), Italian (2.83) and M. australis (4.56) were found highly resistant; nine lines were resistant; 43 lines were moderately resistant and 29 were susceptible to the disease complex. Powdery mildew showed significant positive correlation with Pseudocercospora leaf spot. Highly resistant varieties may be utilized for future disease resistance breeding programme to evolve multiple disease resistant mulberry varieties.     

Role of Phenylalanine Ammonia Lyase and Polyphenol Oxidase in Host Resistance to Bacterial Wilt of Tomato

Plants respond to bacterial pathogen attack by activating various defence responses, which are associated with the accumulation of several factors like defence-related enzymes and inhibitors which serve to prevent pathogen infection. The present study focused on the role of the defence-related enzymes phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO) in imparting resistance to tomato against bacterial wilt pathogen Ralstonia solanacearum . The temporal pattern of induction of these enzymes showed maximum activity at 12 h and 15 h for PAL and PPO, respectively, after the pathogen inoculation (hpi) in resistant cultivars. Twenty different tomato cultivars were analyzed for PAL, PPO and total phenol content following pathogen inoculation. The enzyme activities and total phenol content increased significantly (P < 0.05) in resistant cultivars upon pathogen inoculation. The increase in enzyme activities and total phenol content were not significant in susceptible and highly susceptible cultivars. The role of PAL and PPO in imparting resistance to tomato against bacterial wilt disease is discussed.     

Characterization of disease resistance gene candidates of the nucleotide binding site (NBS) type from banana and correlation of a transcriptional polymorphism with resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">cubense</Emphasis> race 4

Most plant disease resistance (R) genes encode proteins with a nucleotide binding site and leucine-rich repeat structure (NBS-LRR). In this study, degenerate primers were used to amplify genomic NBS-type sequences from wild banana (Musa acuminata ssp. malaccensis) plants resistant to the fungal pathogen Fusarium oxysporum formae specialis (f. sp.) cubense (FOC) race 4. Five different classes of NBS-type sequences were identified and designated as resistance gene candidates (RGCs). The deduced amino acid sequences of the RGCs revealed the presence of motifs characteristic of the majority of known plant NBS-LRR resistance genes. Structural and phylogenetic analyses grouped the banana RGCs within the non-TIR (homology to Toll/interleukin-1 receptors) subclass of NBS sequences. Southern hybridization showed that each banana RGC is present in low copy number. The expression of the RGCs was assessed by RT-PCR in leaf and root tissues of plants resistant or susceptible to FOC race 4. RGC1, 3 and 5 showed a constitutive expression profile in both resistant and susceptible plants whereas no expression was detected for RGC4. Interestingly, RGC2 expression was found to be associated only to FOC race 4 resistant lines. This finding could assist in the identification of a FOC race 4 resistance gene.     

Correlative evidence for peroxidase involvement in disease resistance against <Emphasis Type="Italic">Alternaria</Emphasis> leaf blight of tomato

Eighteen tomato genotypes, with varying degree of response to Alternaria leaf blight disease (ALBD) were used to assess the possible involvement of protease and peroxidase activities in disease response. Pre-infectional protease activity varied noticeably in tested genotypes. Highest pre-infectional protease activity was observed in susceptible genotype CLN-2123. Post-infectional protease activity level was generally lower when compared with pre-infectional level in all genotypes with exception of unchanged level in Tibrido. There was no correlation between post-infectional protease activity and percent disease index (%DI). In contrast, pre- and post-infectional leaf peroxidase activities showed a significant (p < 0.01) negative correlation with %DI. Genotypes with higher pre-infectional peroxidase activity performed better on exposure to Alternaria alternata infection and accumulate enhanced peroxidase activity. Tibrido accumulated highest peroxidase activity while level was lowest in 1621P, which showed highest ALBD incidence. Moreover, genotypes with better resistance to A. alternata infection maintained higher post-infectional peroxidase activity. In resistant (Tibrido) and all moderately resistant genotypes, leaf peroxidase activity raised after inoculation when compared with the pre-inoculation level. I summary, higher pre- and post-infectional peroxidase activity was found to be associated with Alternaria leaf blight resistance. The peroxidase activity can be used as a biochemical tool in marker-assisted screening of tomato germplasm for Alternaria leaf blight resistance.     

Expression of the bacteriophage T4 lysozyme gene in tall fescue confers resistance to gray leaf spot and brown patch diseases

Tall fescue (Festuca arundinacea Schreb.) is an important turf and forage grass species worldwide. Fungal diseases present a major limitation in the maintenance of tall fescue lawns, landscapes, and forage fields. Two severe fungal diseases of tall fescue are brown patch, caused by Rhizoctonia solani, and gray leaf spot, caused by Magnaporthe grisea. These diseases are often major problems of other turfgrass species as well. In efforts to obtain tall fescue plants resistant to these diseases, we introduced the bacteriophage T4 lysozyme gene into tall fescue through Agrobacterium-mediated genetic transformation. In replicated experiments under controlled environments conducive to disease development, 6 of 13 transgenic events showed high resistance to inoculation of a mixture of two M. grisea isolates from tall fescue. Three of these six resistant plants also displayed significant resistance to an R. solani isolate from tall fescue. Thus, we have demonstrated that the bacteriophage T4 lysozyme gene confers resistance to both gray leaf spot and brown patch diseases in transgenic tall fescue plants. The gene may have wide applications in engineered fungal disease resistance in various crops.     

Positional cloning of <Emphasis Type="Italic">ds1</Emphasis>, the target leaf spot resistance gene against <Emphasis Type="Italic">Bipolaris sorghicola</Emphasis> in sorghum

Target leaf spot is one of the major sorghum diseases in southern Japan and caused by a necrotrophic fungus, Bipolaris sorghicola. Sorghum resistance to target leaf spot is controlled by a single recessive gene (ds1). A high-density genetic map of the ds1 locus was constructed with simple sequence repeat markers using progeny from crosses between a sensitive variety, bmr-6, and a resistant one, SIL-05, which allowed the ds1 gene to be genetically located within a 26-kb region on the short arm of sorghum chromosome 5. The sorghum genome annotation database for BTx623, for which the whole genome sequence was recently published, indicated a candidate gene from the Leucine-Rich Repeat Receptor Kinase family in this region. The candidate protein kinase gene was expressed in susceptible plants but was not expressed or was severely reduced in resistant plants. The expression patterns of ds1 gene and the phenotype of target leaf spot resistance were clearly correlated. Genomic sequences of this region in parental varieties showed a deletion in the promoter region of SIL-05 that could cause reduction of gene expression. We also found two ds1 alleles for resistant phenotypes with a stop codon in the coding region. The results shown here strongly suggest that the loss of function or suppression of the ds1 protein kinase gene leads to resistance to target leaf spot in sorghum.     

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.     

Necrotic Lesions as Unusual Symptoms of Ring Rot in the Potato Leaves

Test-tube plants and suspension cell cultures of two cultivars of the potato (Solanum tuberosum L.) differing in their resistance to ring rot caused by Clavibacter michiganensis subsp. sepedonicus and six strains of this bacterium were used to test the relationship between the virulence, the leaf ability to adsorb bacteria, and the symptoms of the disease. In addition to chlorosis and drying, heavy inoculation with virulent strains caused unusual symptoms, such as leaf necrotic lesions. In the resistant cultivar, the necrotic lesions were predominantly local, whereas in the susceptible cultivar, they expanded. Unlike the susceptible cultivar, suspension cells of the resistant cultivar weakly adhered bacteria of the tested strains. Bacteria entered the plants through the leaf stomata. The sorption and penetration were much more pronounced in the susceptible cultivar. It was concluded that strain virulence varies depending on the conditions of inoculation, and uncharacteristic symptoms (necrotic lesions) arise. The local necrotic lesions are considered a hypersensitive response, and exopolysaccharides of the pathogen as the factors of virulence.     

Identification of resistance source in wheat germplasm against spot blotch disease caused by Bipolaris sorokiniana

Four hundred and twenty-two spring wheat germplasm (Triticum aestivum L.) lines belonging to Indian, CIMMYT and Chinese wheat programme were evaluated for their tolerance against natural epiphytotic conditions of spot blotch caused by Bipolaris sorokiniana at the hot spot location, Pusa, Bihar, India. Of the 422 entries screened, none of the genotype showed immunity to the disease, whereas 52 were resistant, 180 moderately susceptible, 171 susceptible and 19 highly susceptible. Indian germplasm lines tended to be more susceptible than lines originated from CIMMYT and China. Chirya 3, Chirya 7 and Mayoor from CIMMYT showed high degree of resistance to the disease both under field and polyhouse conditions. On the basis of the disease severity under field conditions, 20 promising resistant genotypes and 10 highly susceptible lines were isolated for further testing under artificial epiphytotic conditions in polyhouse for genetic analysis and their potential for spot blotch resistance breeding.     

Intercellular chitinase and peroxidase activities associated with resistance conferred by geneLr35to leaf rust of wheat

The effect of leaf rust (Puccinia triticina) infection on intercellular chitinase (EC 3.2.1.14) and peroxidase (EC 1.11.1.7) activities was studied in resistant [RL 6082 (Thatcher/Lr35)] and susceptible (Thatcher) near isogenic wheat (Triticum aestivum L.) lines at seedling, stem elongation and flag leaf stages of plant growth. The levels of activity of these enzymes were low during the seedling and stem elongation stages. Resistant plants at the flag leaf stage, during which the Lr35 resistance gene was maximally expressed, exhibited high constitutive levels of chitinase and peroxidase activities, in contrast to the lower constitutive levels of susceptible plants. The results suggest that chitinase and peroxidase, constitutively present in the intercellular spaces of Thatcher/Lr35 wheat leaves, may play a role in Lr35 mediated resistance to leaf rust.     

Red-Light-Induced Resistance in Broad Bean (Vicia faba L.) to Leaf Spot Disease Caused by Alternaria tenuissima

The effect of different light wavelengths on the development of lesions induced by Alternaria tenuissima in broad bean leaves was investigated. Lesion development was completely suppressed in red‐light‐irradiated broad bean leaflets, irrespective of isolate or spore concentration. Pre‐treatment of leaflets with red light for 24 h before inoculation also suppressed lesion development. Alternaria tenuissima failed to produce infection hyphae in red‐light‐irradiated broad bean leaflets. These results indicate that disease suppression in broad bean leaflets is due to light‐induced resistance. Spore germination fluid (SGF) of A. tenuissima allowed non‐pathogenic Alternaria alternata to infect wounded and unwounded broad bean leaflets kept in the dark, results suggesting that SGF induced susceptibility. Red light suppressed susceptibility induced by A. tenuissima SGF; thus, lesion formation and development were suppressed when leaflets inoculated with the spores of A. alternata suspended in A. tenuissima SGF were kept under red light. From these results, we conclude that red light induced resistance in broad bean to leaf spot disease caused by A. tenuissima, and that SGF induced susceptibility of broad bean leaflets to a non‐pathogenic isolate of A. alternata.