Transgenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens

Defensins are small positively charged, antimicrobial peptides (~5 kDa in size) and some of them exhibit potent antifungal activity. We have cloned the complete cDNA containing an ORF of 243 bp of a defensin of mustard. The deduced amino acid sequence of the peptide showed more than 90% identity to the amino acid sequence of the well-characterized defensins, RsAFP-1 and RsAFP-2 of Raphanus sativus. We have generated and characterized transgenic tobacco and peanut plants constitutively expressing the mustard defensin. Transgenic tobacco plants were resistant to the fungal pathogens, Fusarium moniliforme and Phytophthora parasitica pv. nicotianae. Transgenic peanut plants showed enhanced resistance against the pathogens, Pheaoisariopsis personata and Cercospora arachidicola, which jointly cause serious late leaf spot disease. These observations indicate that the mustard defensin gene can be deployed for deriving fungal disease resistance in transgenic crops.     

Increased resistance to late leaf spot disease in transgenic peanut using a combination of PR genes

Peanut (Arachis hypogaea L.) is the sixth most important oil seed crop in the world. Yield loss due to Cercospora leaf spot (early and late leaf spots) is a serious problem in cultivating this crop. Non-availability of resistant genes within crossable germplasms of peanut necessitates the use of a genetic engineering strategy to develop genetic resistance against various biotic stresses. The pathogenesis-related (PR) proteins are a group of plant proteins that are toxic to invading fungal pathogens, but are present in trace amounts in plants. The PR proteins, PR-5 and defensins, are potent antifungal proteins. A double gene construct with SniOLP (Solanum nigrum osmotin-like protein) and Rs-AFP2 (Raphanus sativus antifungal protein-2) genes under separate constitutive 35S promoters was used to transform peanut plants. Transgenic peanut plants expressing the SniOLP and Rs-AFP2 genes showed enhanced disease resistance to late leaf spot based on a reduction in number and size of lesions on leaves and delay in the onset of Phaeoisariopsis personata leaf spot disease. PCR, RT–PCR, and Southern hybridization analyses confirmed stable integration and expression of these genes in peanut transgenics. The results demonstrate the potential of SniOLP and Rs-AFP2 genes in developing late leaf spot disease resistance in transgenic peanut.     

Dothistromin as a metabolite of Cercospora arachidicola

The known phytotoxin dothistromin has been newly identified as a metabolite of the peanut pathogen Cercospora arachidicola. The potential of the substance as a mycotoxin is discussed.     

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.     

Biphasic diurnal cycle of ascus development of Taphrina maculans Butler

Taphrina maculans Butler inciting the brown leaf spot disease of turmeric (Curcuma longa L.) produces cuboid ascogenous cells several layers in depth in the subcuticular interspaces of the epidermis. The ascogenous cells germinate to produce asci centrifugally maturing toward the periphery of the stroma, expelling octosporous microcolonies of asco-blastospores on the leaf surface inciting secondary infection in favorable environment. Occurrence of a rhythmic cycle of ascus development and ascospore discharge giving 2 peaks of ascospore discharge each day has been demonstrated in Taphrina maculans. The cycle is directly affected by atmospheric temperature, availability of free moisture on the leaf surface and sunlight. Free moisture on a leaf surface appropriately soaks the infection spots (ascogenous cells) and induces ascus elongation and ascospore discharge, when a suitable atmospheric temperature is reached. Sunlight may adversely affect the cycle by increasing the temperature and lowering humidity in the atmosphere.     

Ethylene Production and Leaflet Abscission of Three Peanut Genotypes Infected with Cercospora arachidicola Hori

Ethylene can induce abscission of leaves and other plant organs. Increased ethylene production by plant tissues can occur after invasion by microorganisms. The fungus Cercospora arachidicola Hori, attacks peanut leaflets and causes defoliation. Our objective was to determine if ethylene was involved in this defoliation. Leaves of three peanut, Arachis sp., genotypes were inoculated with C. arachidicola. Two genotypes, `Tamnut 74' and PI 109839, produced ethylene and were defoliated. The third genotype, PI 276233, a wild species, did not produce ethylene above control levels and was not defoliated. Increase in ethylene production by Tamnut 74 and PI 109839 coincided with appearance of disease symptoms. Tamnut 74 produced the most ethylene, but PI 109839 was equally defoliated. Thus, less overall ethylene production did not necessarily indicate a more resistant genotype in this system unless ethylene production remained at control levels, as it did for PI 276233. Ethylene sufficient to initiate abscission could have been produced by the seventh day after inoculation when it was similar for both Tamnut 74 and PI 109839, but 3 to 4 times control amounts. This occurred before the rapid increase in ethylene production and before disease symptoms were visible. Silver ion, a potent inhibitor of ethylene action, was sprayed at three concentrations on intact Tamnut 74 plants. All rates reduced abscission and 150 mg/liter Ag(I) decreased abscission to below 10%. The data indicate that ethylene produced by peanut leaves in response to C. arachidicola infection initiates abscission and that ethylene action can be blocked by Ag(I) in such a host-pathogen interaction.     

Incidence and severity of frogeye leaf spot and associated yield losses in soybeans in agroecological zone II of Zambia

The incidence and severity of frogeye leaf spot of soybean (Glycine max (L.) Merr.) was studied in agroecological region II of Zambia during the 1997/98 crop growing season. A survey was conducted on farmers' fields on SCSI Kaleya, Magoye and Hernon-147 cultivars. Disease incidence and severity was assessed by monitoring disease increments at two weeks interval (beginning of January to April) from nine fields, three from each province. Soybean cultivars were evaluated for yield losses resulting from frogeye leaf spot. Field plots of each cultivar were either sprayed twice with benomyl (benlate) or not sprayed at all. The results showed that the incidence of frogeye leaf spot was highest in Southern province (5.1), followed by Lusaka province (4.9) while Central province had the lowest disease incidence (1.8). Values for area under disease progress curve (AUDPC) were significantly greater (P < 0.05) for Lusaka and Southern provinces than for Central province. Yields in benomyl protected plots ranged from 1444 kg ha⁻¹ to 2320 kg ha⁻¹ and were significantly different among the cultivars. Average yields of non protected plants were reduced by 30.5% for Kaleya, 35.6% for Hernon-147 and 37.2% for SCS1. Incidence and severity increased with time and varied depending on weather parameters and susceptibility of cultivars to the disease. Yield losses due to frogeye leaf spot occurred through a reduction in seed size. Differences in weather conditions and amount of inocula are believed to contribute to the observed variation in incidence and severity of the disease at different locations. This revised version was published online in July 2006 with corrections to the Cover Date.     

Overexpression of a chitinase gene in transgenic peanut confers enhanced resistance to major soil borne and foliar fungal pathogens

A chitinase gene from rice (Rchit) was introduced into three varieties of peanut through Agrobacterium-mediated genetic transformation resulting in 30 transgenic events harboring the Rchit gene. Stable integration and expression of the transgenes were confirmed using PCR, RT-PCR and Southern blot analysis. Progeny derived from selfing of the primary transgenic events revealed a Mendelian inheritance pattern (3:1) for the transgenes. The chitinase activity in the leaves of the transgenic events was 2 to 14-fold greater than that in the non-transformed control plants. Seeds of most transgenic events showed 0–10 % A. flavus infection during in vitro seed inoculation bioassays. Transgenic peanut plants evaluated for resistance against late leaf spot (LLS) and rust using detached leaf assays showed longer incubation, latent period and lower infection frequencies when compared to their non-transformed counterparts. A significant negative correlation existed between the chitinase activity and the frequency of infection to the three tested pathogens. Three progenies from two transgenic events displayed significantly higher disease resistance for LLS, rust and A. flavus infection and are being advanced for further evaluations under confined field conditions to confirm as sources to develop peanut varieties with enhanced resistance to these fungal pathogens.     

Biology and Control of Entyloma eryngii (Cda.) de By., the Cause of a Leaf Spot Disease on the Blue Thistle (Eryngium alpinum L.)

The blue thistle (Eryngium alpinum L.), an indigenous perennial flower of the alps, is grown in mountainous regions of south-eastern Switzerland. The commercial value of the fresh cut flowers can be seriously reduced due to a leaf spot disease caused by the smut fungus Entyloma eryngii (Cda.) de By. The fungus produces thick walled chlamydospores and two types of conidia. The disease appears to be favoured by cool-rainy weather and a dense foilage resulting in long periods of leaf wetness. Control of the disease can be achieved with the protective fungicide mancozeb or even more efficiently with the systemic ergosterol-biosynthesis-inhibiting fungicides cyproconazole and difenoconazole.     

Disease assessment methods and their use in simulating growth and yield of peanut crops affected by leafspot disease

Peanut (Arachis hypogaea) crops in Benin often experience late leafspot (Cercosporidium personatum), which causes severe yield losses associated with leaf defoliation and necrosis. The objective of this research was to determine the best method of disease assessment and to test its utility in the CROPGRO‐peanut model to simulate growth and yield as affected by late leafspot in early and late maturing peanut cultivars grown at different sowing dates under rain‐fed conditions (without irrigation) in northern Benin. Two peanut cultivars TS 32‐1 and 69–101 were sown on three dates between May and August during 1998 and 1999. In both years there was severe occurrence of late leafspot and the progression of disease was earlier and faster with later sowing dates. Overall, the long duration cultivar 69–101 produced greater yield than the short duration cultivar TS 32‐1. The CROPGRO‐peanut model was able to predict and simulate the observed crop and pod dry matter over time when input on percent diseased leaf area and percent defoliation were provided. Of several disease assessments, the best approach was to input measured percent main‐stem defoliation above the fourth node and percent diseased leaf area estimated from visual leafspot score.     

Fungal foliar diseases of Rauwolfia serpentina in wild,its seasonal occurrence,seed transmission and disease management

Rauwolfia serpentina is an important medicinal herb. Studies were conducted to determine causal organisms of foliar diseases in R. serpentina in Bhadra Wildlife Sanctuary during 2006–2009. The foliar disease incidence and its distribution and disease severity in nine state forest regions of the sanctuary were determined. The seedborne nature and transmission of the causal organism was also determined. The management of seedborne inoculum was done by seed dressing with fungicides. The effect of foliage infection on secondary metabolite content was also determined. Results of the present study indicated that Cercospora rauwolfiae is major leaf spot disease causing pathogen. The disease is homogeneously distributed through the study area. The foliar disease severity was high in Kagemanegiri forest during October–November. The minor leaf spot disease caused by Alternaria alternata occurred occasionally. Alternaria alternata is seedborne and seed transmitted and could be managed by seed treatment with Captra or Hyzeb. The secondary metabolites like alkaloids and steroids decreased with increase in foliar infection by C. rauwolfiae, while phenol and flavonoid contents increased. The study suggested that R. serpentina is affected in wild by C. rauwolfiae and A. alternata. The latter pathogen is seedborne and seed transmitted and controlled by seed treatment.     

Soil moisture mediated interaction between <Emphasis Type="Italic">Polygonatum biflorum</Emphasis> and leaf spot disease

Fungal pathogens can regulate the abundance and distribution of natural plant populations by inhibiting the growth, survival, and reproduction of their hosts. The abiotic environment is a crucial component in host–pathogen interactions in natural plant populations as favorable conditions drive pathogen development, reproduction, and persistence. Foliar plant pathogens, such as fungal lesions referred to generically as “leaf spot disease,” are particularly responsive to increased moisture levels, but the manner in which the abiotic environment drives disease dynamics, and how these diseases regulate natural plant populations, is not fully understood. We investigate (1) the impact of ambient soil moisture and diffuse light on the prevalence of a leaf spot pathogen (Phyllosticta sp.) in a natural population of Polygonatum biflorum, an understory herb native to deciduous forest understories in the eastern US, and (2) the effects of the fungal pathogen on the survival, growth, and abundance of the plants. We tracked six P. biflorum populations and disease incidence, as well as soil moisture and diffuse light, between 2003 and 2005 in the understory deciduous forest of the southern Appalachian Mountains, North Carolina, USA. Results show that both the occurrence of P. biflorum and the prevalence of P. biflorum leaf spot disease are highest where soil moisture is intermediate and diffuse light is lowest. Disease occurrence depends upon plant presence, but it also adversely impacts plant survival, abundance, and growth. These results suggest that leaf spot disease is likely to impact population dynamics, which in turn vary as a function of environmental drivers.     

Effects of light leaf spot (Pyrenopeziza brassicae) on yield of winter oilseed rape (Brassica napus)

The relationship between development of light leaf spot and yield loss in winter oilseed rape was analysed, initially using data from three experiments at sites near Aberdeen in Scotland in the seasons 1991/92, 1992/93 and 1993/94, respectively. Over the three seasons, single-point models relating yield to light leaf spot incidence (% plants with leaves with light leaf spot) at GS 3.3 (flower buds visible) generally accounted for more of the variance than single-point models at earlier or later growth stages. Only in 1992/93, when a severe light leaf spot epidemic developed on leaves early in the season, did the single-point model for disease severity on leaves at GS 3.5/4.0 account for more of the variance than that for disease incidence at GS 3.3. In 1991/92 and 1992/3, when reasonably severe epidemics developed on stems, the single-point model for light leaf spot incidence (stems) at GS 6.3 accounted for as much of the variance. Two-point (disease severity at GS 3.3 and GS 4.0) and AUDPC models (disease incidence/severity) accounted for more of the variance than the single-point model based on disease incidence at GS 3.3 in 1992/93 but not in the other two seasons. Therefore, a simple model using the light leaf spot incidence at GS 3.3 (x) as the explanatory variable was selected as a predictive model to estimate % yield loss (y_r): y_r= 0.32x– 0.57. This model fitted all three data sets from Scotland, When data sets from Rothamsted, Rosemaund and Thurloxton in England were used to test it, this single-point predictive model generally fitted the data well, except when yield loss was clearly not related to occurrence of light leaf spot. However, the regression lines relating observed yield loss to light leaf spot incidence at GS 3.3 often had smaller slopes than the line produce, by the model based on Scottish data.     

Factors affecting bacterial black spot of mangoes caused by Xanthomonas campestris pv. mangiferaeindicae

Bacterial black spot of mango caused by Xanthomonas campestris by. mangiferaeindicae was found to be essentially a wound pathogen of leaves under glasshouse conditions, with maximum infection occurring at a temperature regime of 22/26 ^oC (night/day). There were significant differences in leaf susceptibility among cultivars, cv. Sensation being less, and cv. Haden more susceptible than cvs Zill and Kent, which agrees with field observations of fruit infection. The organism is a phylloplane resident year round in the field and inoculum levels in the tree canopy affect ultimate fruit infection. The major weather factor affecting fruit infection was rainfall which showed a significant correlation (r = 0–77) with levels of infection after allowing for an approximate 2 wk latent period. Other significant correlations were with a wind/rain index (r = 0–73) and a storm index (r = 0–66). The correlation of disease with the duration of free water was non-significant (r = 0–22).     

Components of resistance to Cercospora arachidicola and Cercosporidium personatum in groundnuts*

The reactions of four groundnut cultivars to inoculation with two leaf spot pathogens (Cercospora arachidicola and Cercospodirium personatum) were investigated. Resistant cultivars showed longer latent periods, reduced sporulation and less defoliation; however, none of the cultivars was resistant to both pathogens. Numbers of lesions, time to leaflet loss and the percentage of spores which produced lesions were influenced by the concentration of C. personatum inoculum applied to the leaves and there were significant cultivar differences in lesion numbers at the lowest inoculum level. Only the first two characters were affected by applied spore concentration in the study with C. arachidicola. Generally it was found that lower inoculum levels gave a better separation of cultivars. The level of resistance was similar to that found in the slow resting reaction of cereal crops and should be useful in a groundnut breeding programme.     

Rhizobium radiobacter conjugation and callus-independent shoot regeneration used to introduce the cercosporin export gene cfp from Cercospora into sugar beet (Beta vulgaris L.)

Leaf spot disease caused by Cercospora is responsible for crop and profitability losses in sugar beet crops in the US and worldwide. The cfp gene that encodes a protein that exports phytotoxic cercosporins from Cercospora was conjugally transferred to sugar beet using Rhizobium radiobacter (Agrobacterium tumefaciens), to improve Cercospora-induced leafspot resistance. Conditions for shoot regeneration were optimized to increase regeneration/transformation efficiencies. Low-light and room-temperature conditions were favorable to sugar beet regeneration without callus when cytokinin had been added to the tissue culture medium. Using this procedure adventitious shoots from leaf pieces were obtained in a simple, one-step regeneration procedure. T7, a cfp-transgenic clone verified by PCR with gene-specific primers, is being propagated for leaf spot disease resistance evaluation.     

Genetic variability in mulberry against Myrothecium leaf spot (Myrothecium roridum) and identification of resistance genotypes

Disease response of 30 mulberry genotypes to Myrothecium leaf spot (Myrothecium roridum) was studied under inoculated condition. It was observed that 10 genotypes were resistant, 16 genotypes moderately resistant and 4 genotypes moderately susceptible to the disease. Area under disease progress curve (AUDPC) and apparent infection rate was found significantly lower in the resistant genotypes. Correlation study revealed that percent disease index (PDI) has significant positive correlation with AUDPC and apparent infection rate. Genetic analysis of disease-resistant traits (PDI and mean AUDPC) revealed that phenotypic coefficient of variation (PCV) was higher than genotypic coefficient of variation (GCV) for both PDI and AUDPC, and GCV/PCV ratio was also found high which indicated that disease-resistant traits to Myrothecium leaf spot were not much influenced by environment. High heritability coupled with high genetic advance indicated that the disease-resistant traits are due to additive gene effect thereby indicating the amenability of disease-resistant in the selection process. Hence disease-resistant mulberry genotypes viz. C-763, S-34, Jodhpur, Cyprus, Australian and Hungarian may be used as source of resistance to Myrothecium leaf spot for future breeding programme. Besides, high yielding genotypes viz. Tr-10, C-763 and S-34 may be recommended for commercial exploitation     

Environmental factors impact temporal Passalora sequoiae conidia counts from Leyland cypress

Needle blight disease, caused by Passalora sequoiae, results in a progressive loss of leaf tissue on Leyland cypress (×Cupressocyparis leylandii) within container and field tree nurseries, in the landscape and on Christmas tree farms. Fungicide schedules have been developed in response to seasonal symptom progression and conidia dispersion without clarification of the influence of weather conditions on disease. Conidia count data of P. sequoiae were collected approximately weekly from June to December in 2001 in Watkinsville, GA, and in 2002 in Dearing, GA. Peak conidia numbers were trapped in September and October. Daily weather data summaries were obtained from a weather station at each location. Deterministic models were developed individually for summer (1 June to 30 September) and fall (1 October to 21 December) seasons. A three‐day moving average (MA) of average daily temperature (avgT) had a positive quadratic relationship with conidia counts in the summer season. A 10‐day MA of avgT, vapour pressure deficit (VPD) and total solar radiation (SolR) had a positive quadratic, a negative linear and positive linear relationship, respectively, with conidia counts in the fall season. The models show that the seasonal shift to cooler fall temperatures, along with lower VPD and SolR, favour production and dispersal of P. sequoiae conidia.     

Identification of phytoplasmas associated with sesame phyllody disease in southeastern Iran

Phyllody disease is a threat to sesame production in Kerman province, southeastern Iran. RFLP analysis of PCR products of phytoplasma-specific 16S rRNA gene (1.8 kb) and phylogenetic analyses of 16S-23S rDNA spacer region (SR) sequence indicated that the predominant agent associated with sesame phyllody in Kerman province is a phytoplasma with 100% similarity with eggplant big bud, and peanut witches’-broom phytoplasmas, members of “Candidatus Phytoplasma aurantifolia” from Iran and China, respectively. Among the samples tested, only one strain (SPhSr1), had a unique RFLP profile and its SR was 100% similar in nucleotide sequence with the phytoplasma carried by Orosius albicinctus and Helianthus annus witches’-broom phytoplasma from Iran, members of “Ca. Phytoplasma trifolii”. Virtual RFLP patterns of SPhJ2 (representative of the predominant PCR-RFLP profiles) SR sequence were identical to those of peanut witches’-broom phytoplasma (16SrII-A, JX871467). However, SPhSr1 SR sequence patterns resemble (99.7%) those of vinca virescence phytoplasma (16SrVI-A, AY500817).     

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.