Overexpression of citrus polygalacturonase-inhibiting protein in citrus black rot pathogen Alternaria citri

The rough lemon (Citrus jambhiri) gene encoding polygalacturonase-inhibiting protein (RlemPGIPA) was overexpressed in the pathogenic fungus Alternaria citri. The overexpression mutant AcOPI6 retained the ability to utilize pectin as a sole carbon source, and the overexpression of polygalacturonase-inhibiting protein did not have any effect on the growth of AcOPI6 in potato dextrose and pectin medium. The pathogenicity of AcOPI6 to cause a black rot symptom in citrus fruits was also unchanged. Polygalacturonase-inhibiting protein was secreted together with endopolygalacturonase into culture filtrates of AcOPI6, and oligogalacturonides were digested from polygalacturonic acid by both proteins in the culture filtrates. The reaction mixture containing oligogalacturonides possessed activity for induction of defense-related gene, RlemLOX, in rough lemon leaves.     

Arbuscular mycorrhiza reduces susceptibility of tomato to Alternaria solani

Mycorrhiza frequently leads to the control of root pathogens, but appears to have the opposite effect on leaf pathogens. In this study, we studied mycorrhizal effects on the development of early blight in tomato (Solanum lycopersicum) caused by the necrotrophic fungus Alternaria solani. Alternaria-induced necrosis and chlorosis of all leaves were studied in mycorrhizal and non-mycorrhizal plants over time course and at different soil P levels. Mycorrhizal tomato plants had significantly less A. solani symptoms than non-mycorrhizal plants, but neither plant growth nor phosphate uptake was enhanced by mycorrhizas. An increased P supply had no effect on disease severity in non-mycorrhizal plants, but led to a higher disease severity in mycorrhizal plants. This was parallel to a P-supply-induced reduction in mycorrhiza formation. The protective effect of mycorrhizas towards development of A. solani has some parallels to induced systemic resistance, mediated by rhizobacteria: both biocontrol agents are root-associated organisms and both are effective against necrotrophic pathogens. The possible mechanisms involved are discussed.     

Effect of soil-applied NPK fertilizers on severity of black spot disease (Alternaria brassicae) and yield of oilseed rape

Effect of soil application of eight combinations of NPK fertilizers on the severity of black spot disease (BSD), caused by Alternaria brassicae (Sacc.) Berk., and yield of short duration oilseed rape (Brassica campestris L) were investigated under both pot and field conditions in 1987–88, 1988–89 and 1990–91. The severity of BSD was significantly greater (36–48%) on plants grown in ground treated with NP (N 90 kg ha^–1+P 40 kg ha^–1) applied as urea and single superphosphate respectively than on plants from the unfertilized control (NoPoKo) (o). However, the severity of BSD was significantly smaller (25–33%) when K (40 kg ha^–1) was applied as muriate of potash than in plants from control and NP treatments. The effect of NK (N 90 kg ha^–1+K 40 kg ha^–1) in decreasing the severity of BSD was increasingly more pronounced than the effects of PK (P 40 kg ha^–1+K 40 kg ha^–1), NP and K (40 kg ha^–1) applications. The decrease in the severity of BSD due to K was due to increased production in plants of phenolics which inhibited conidial germination and decreased sporulation of A. brassicae.The decrease in the severity of BSD due to NK application gave consistently increased seed yield 68% more than those of control and other treatments. The K-fertilized plants also showed increased resistance to lodging, increased 1000-seed weight and decreased seed infection. Seeds obtained from K-fertilized plants showed good seed germinability and vigorous seeding growth.     

Patulin and aflatoxin in brown rot lesion of apple fruits and their regulation

Aspergillus flavus, A. niger, Penicillium expansum and Rhizopus stolonifer were the most frequently isolated fungi from healthy apple fruits. Alternaria alternata was the most common organism of rotten apple fruits, followed by A. niger, A. flavus, P. expansum and R. stolonifer. The prevalent type of decay, brown rot lesion, is caused by R. stolonifer followed by A. flavus, A. niger, A. alternata and P. expansum. Sodium hypochlorite had good curative properties against fruit rots. The main natural mycotoxins produced in rotten apple were patulin and aflatoxins. The optimum temperature for patulin production by P. expansum was 15 °C after 15 days. Complete inhibition of patulin formation was attained using 0.2% lemon oil and > 90% inhibition using 0.05% lemon and 0.2% orange oils. Also significant inhibition (> 90%) of aflatoxin production was observed with 0.2% lemon oil. This revised version was published online in July 2006 with corrections to the Cover Date.     

Tomato resistance to Alternaria stem canker: localization in host genotypes and functional expression compared to non-host resistance

Summary The Alternaria stem canker resistance locus (Asc-locus), involved in resistance to the fungal pathogen Alternaria alternata f. sp. lycopersici and in insensitivity to host-specific toxins (AAL-toxins) produced by the pathogen, was genetically mapped on the tomato genome. Susceptibility and resistance were assayed by testing a segregating F₂ population for sensitivity to AAL-toxins in leaf bioassays. Linkage was observed to phenotypic markers solanifolium and sunny, both on chromosome 3. For the Asc-locus, a distance of 18 centiMorgan to solanifolium was calculated, corresponding to position 93 on chromosome 3. This map position of the resistance locus turned out to be the same in three different resistant tomato accessions, one Dutch and two American, that are at least 40 years apart. AAL-toxin sensitivity in susceptible and resistant tomato genotypes was compared with AAL-toxin sensitivity in a non-host Nicotiana tabacum during different levels of plant cell development. In susceptible and resistant tomato genotypes, inhibitory effects were demonstrated at all levels, except for leaves of resistant genotypes. However, during pollen and root development, inhibitory effects on susceptible genotypes were larger than on resistant genotypes. In the non-host Nicotiana tabacum, hardly any effects of AAL-toxins were demonstrated. Apparently, a cellular target site is present in tomato, but not in Nicotiana tabacum. It was concluded that three levels of AAL-toxin sensitivity exist: (1) a susceptible host sensitivity, (2) a resistant host sensitivity, (3) a non-host sensitivity, and that the resistance mechanism operating in tomato is different from that operating in Nicotiana tabacum.     

Cytokinin-like substances and accumulation of labelled metabolites at infection sites of Alternaria brassicicola

Green islands were observed on mustard leaves beneath the infection drops containing germinating conidia of Alternaria brassicicola, when the surrounding uninfected tissue had yellowed due to senescence. Green island formation has been correlated with the secretion of cytokinin-like substances by the pathogens. A. brassicicola secreted cytokinin-like substances in a liquid synthetic medium and their application to detached host leaves evoked the formation of green islands in the dark. ¹⁴C studies confirmed that green islands act as metabolic sinks in which photosynthates are retained or accumulated. Cytokinin-like substances appear to be actively involved in infection and pathogenesis of A. brassicicola.     

FW2.2 and cell cycle control in developing tomato fruit: a possible example of gene co-option in the evolution of a novel organ

fw2.2 is one of the few QTLs thus far isolated from plants and the first one known to control fruit size. While it has been established that FW2.2 is a regulator (either directly or indirectly) of cell division, FW2.2 does not share sequence homology to any protein of known function (Frary et al. Science 289:85–88, 2000; Cong et al. Proc Natl Acad Sci USA 99:13606–13611, 2002; Liu et al. Plant Physiol 132:292–299, 2003). Thus, the mechanism by which FW2.2 mediates cell division in developing fruit is currently unknown. In an effort to remedy this situation, a combination of yeast two-hybrid screens, in vitro binding assays and cell bombardment studies were performed. The results provide strong evidence that FW2.2 physically interacts at or near the plasma membrane with the regulatory (beta) subunit of a CKII kinase. CKII kinases are well-studied in both yeast and animals where they form part of cell cycle related signaling pathway. Thus while FW2.2 is a plant-specific protein and regulates cell division in a specialized plant organ (fruit), it appears to participate in a cell-cycle control signal transduction pathway that predates the divergence of single- and multi-cellular organisms. These results thus provide a glimpse into how ancient and conserved regulatory processes can be co-opted in the evolution of novel organs such as fruit.     

Wound-induced changes in tomato leaves and their effects on the feeding patterns of larval lepidoptera

Several studies have shown changes in the patterns of damage from feeding insects associated with changes in palatability and overall consumption as a result of wound-induced chemical changes in plants. This paper describes how the pattern of feeding damage made by the larvae of Spodoptera littoralis Boisd. (Lepidoptera: Noctuidae) on tomato is affected by changes in palatability of the leaves. Two sorts of responses to leaves from plants that had received prior damage were observed. Larvae offered a choice of leaves tended to take fewer meals on leaves from previously-wounded plants than on control leaves, frequently rejecting the former after sampling them. On wounded plants this rejection behaviour was associated with a shift in feeding site towards the base of the plant. However, starved larvae offered only a single excised leaf readily ate leaves from wounded plants but took shorter meals on these leaves than on controls. Although it was not directly tested it is possible that this difference in response reflected changes in food selectivity with a differing level of satiation. The results are considered in relation to the adaptive significance of the plant of changes in within-plant distributions of herbivore damage.     

Interacting effects of time,temperature, pH and simple sugars on biomass and toxic metabolite production by three Alternaria spp.

Biomass and toxic metabolite production by Altemaria hemicota, A. solani and A. tenuis in modified Czapek Dox broth (mCDB) as affected by incubation time (up to 4 wk) temperature (6, 15 and 25 °C), pH (3.0, 5.0 and 8.0) and sugars (glucose, fructose and sucrose) was studied. Biomass production was greatest at 25 ° C and pH 8.0 but toxin production was enhanced at 15 °C. In general, biomass production was promoted by up to 9% glucose and sucrose and 6% fructose; however, toxin production was simultaneously reduced by elevated sugars in mCDB over a 4-wk incubation period.     

The effect of temperature on salt uptake by tomato plants with diurnal and noctural waterlogging of salinized rootzones

Summary Tomato plants were grown at three constant temperatures (10, 20 and 28°C) with drained or waterlogged rootzones and were irrigated with saline solution (0.09M NaCl).Each increase in temperature resulted in an increase in leaf Na-ion and Cl-ion concentrations in plants grown with drained rootzones. However, with plants grown with waterlogged rootzones, maximum leaf concentrations of Na-ions and Cl-ions occurred at 20°C.At 10°C there were no differences between Na-ion and Cl-ion concentrations for drained or waterlogged treatments. At 20 and 28°C, waterlogging of the rootzone resulted in significantly higher concentrations of Na-ions and Cl-ions in leaf and stem tissues than occurred with drained rootzones.There were no differences in Na-ions and Cl-ions and Cl-ions in plant tops if plants were waterlogged with saline solution during the day or night.Transpiration increased significantly with each increase in temperature but showed no other treatment dependent responses.     

Conditions for fruit body formation in the white rot basidiomycete Phanerochaete chrysosporium

In Phanerochaete chrysosporium fruit body formations is subject to strong catabolite repression by glucose in the presence of physiological levels of nitrogen. Walseth cellulose was found to be the best source of carbon for the induction of fruit body and consequent basidiospore synthesis. Ejected basidiospores collected from cultures grown under these conditions for two weeks are contaminated with neither conidia nor mycelial fragments and are therefore suitable for genetic analysis of recombination. Under conditions of nitrogen limitation, the glucose catabolite repression of fruit body synthesis was relieved. Exogenous adenosine 3,5-monophosphate but not other related nucleotides, also relieved glucose catabolite repression of fruit body formation.     

Evaluation of some fungicides for seed treatment and foliar application in management of damping-off of seedlings and blight of rapeseed caused by Alternaria brassicae

Eighteen fungicides were first evaluated for their effects on growth of Alternaria brassicae and for ascertaining their fungicidal and fungistatic natures in artificial cultures. The chemicals emerging fungicidal in action, were later evaluated for their efficacy as seed treatment and foliar application in the management of damping-off of seedlings and blight of rapeseed separately. Of 18 fungicides tested, six fungicides, viz., Dithane M-45, Dithane Z-78, Ziram, Difolatan-80, Blitox-50 and Benlate completely inhibited the growth of the pathogen and were fungicidal in action. Thiram and Brestan-60, which also caused total growth inhibition, were, however, fungistatic. Benlate (0.1 %) followed by Dithane M-45 was best seed-dressing fungicide for controlling damping-off of seedlings. Dithane M-45 (0.2%) followed by Dithane Z-78 as foliar spray was most effective for controlling the blight and increasing the yield in field trials.     

The effect of nitrogen fertilizer and irrigation on black point incidence in soft white spring wheat

Agronomic studies were conducted to examine the effect of fertilizer N on black point incidence in Fielder soft white spring wheat (Triticum aestivum L. em Thell.). Black point incidence rose with increases in the amount of N supplied either as fertilizer applied during the growing season in irrigation water or as soil N, specifically nitrate, from fertilizer N application in previous years. A comparison of four different irrigation regimes demonstrated that black point incidence was highest under frequent irrigation (irrigate to field capacity at 75% available moisture) and lowest under conventional irrigation (irrigate to field capacity at 50% available soil moisture). In each irrigation regime, disease incidence increased as N rates were raised from 0 to 120 kg ha^-1. A residual fertilizer-N study demonstrated in 1985 and 1986 that black point incidence generally rose with increasing levels of nitrogen from either preplant applications in the spring or soil nitrate from the previous year. However, additions of fertilizer N were shown to slightly reduce black point incidence at soil nitrate levels above 150 kg ha^-1. A two-year fertilizer N study demonstrated that in treatments receiving the same amount (90 kg ha^-1) of fertilizer N, the amount broadcast as a preplant treatment versus the amount applied in irrigation water in a fertigation treatment had no effect on black point incidence, but all fertilized treatments had significantly higher levels of disease than the unfertilized check.Contribution no. 3879016     

Negative regulation of <Emphasis Type="Italic">KNOX</Emphasis> expression in tomato leaves

Leaves of seed plants can be described as simple, where the leaf blade is entire, or dissected, where the blade is divided into distinct leaflets. Both simple and dissected leaves are initiated at the flanks of a pluripotent structure termed the shoot apical meristem (SAM). In simple-leafed species, expression of class I KNOTTED1-like homeobox (KNOX) proteins is confined to the meristem while in many dissected leaf plants, including tomato, KNOX expression persists in leaf primordia. Elevation of KNOX expression in tomato leaves can result in increased leaflet number, indicating that tight regulation of KNOX expression may help define the degree of leaf dissection in this species. To test this hypothesis and understand the mechanisms controlling leaf dissection in tomato, we studied the clausa (clau) and tripinnate (tp) mutants both of which condition increased leaflet number phenotypes. We show that TRIPINNATE and CLAUSA act together, to restrict the expression level and domain of the KNOX genes Tkn1 and LeT6/Tkn2 during tomato leaf development. Because loss of CLAU or TP activity results in increased KNOX expression predominantly on the adaxial (upper) leaf domain, our observations indicate that CLAU and TP may participate in a domain-specific KNOX repressive system that delimits the ability of the tomato leaf to generate leaflets.     

Transgenic Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) expressing tomato glucanase leads to arrested growth of <Emphasis Type="Italic">Alternaria brassicae</Emphasis>

Brassica juncea is an important oilseed crop of the Indian sub-continent. Yield loss due to fungal disease alternaria leaf spot caused by Alternaria brassicae is a serious problem in cultivation of this crop. Nonavailability of resistance genes within crossable germplasms of Brassica necessitates use of genetic engineering strategies to develop genetic resistance against this pathogen. The pathogenesis related (PR) proteins are group of plant proteins that are toxic to invading fungal pathogens, but are present in plant in trace amount. Thus, overexpression of PR proteins leads to increased resistance to pathogenic fungi in several crops. The PR protein glucanase hydrolyzes a major cell-wall component, glucan, of pathogenic fungi and acts as a plant defense barrier. We report the expression of a class I basic glucanase gene, under the control of CaMV 35S promoter, in Indian mustard and its genetic resistance against alternaria leaf spot. Southern and Northern hybridization confirmed stable integration and expression of the glucanase gene in mustard transgenics. Several independent transgenics were screened in vitro and under poly house conditions for their resistance against Alternaria brassicae. In an in vitro antifungal assay, transgenics arrested hyphal growth of Alternaria brassicae by 15-54%. Under pathogen-challenged conditions in poly house, the transgenics showed restricted number, size and spread of lesions caused by Alternaria brassicae. Also, the onset of disease was delayed in transgenics compared to untransformed parent plants. The results demonstrate potentiality of a PR protein from a heterologous source in developing alternaria leaf spot resistance in Indian mustard.     

Effect of radiation dose on the production of and the extent of asymmetry in tomato asymmetric somatic hybrids

Summary Asymmetric somatic hybrids were recovered following fusion of tomato leaf mesophyll protoplasts with irradiated protoplasts isolated from Lycopersicon pennellii suspension cells. The asymmetry was determined by scoring the regenerants at between 20 and 24 loci using isozymes and restriction fragment length polymorphisms. In addition, three quantitative traits, fruit size, leaf shape, and stigma exsertion, were measured in the regenerants. The recovery of asymmetric somatic hybrids was as high as 50% of the regenerants, and there was no requirement for the transfer of a selectable marker gene from the irradiated partner. The amount of nuclear DNA transferred from the irradiated protoplast fusion partner was found to be inversely proportional to the radiation dose. It was possible to recover tomato asymmetric somatic hybrids which were self-fertile and contained limited amounts of genetic information from L. pennelli.     

Ripening-related occurrence of phosphoenolpyruvate carboxykinase in tomato fruit

Phosphoenolpyruvate carboxykinase (PEPCK) is present in ripening tomato fruits. A cDNA encoding PEPCK was identified from a PCR-based screen of a cDNA library from ripe tomato fruit. The sequence of the tomato PEPCK cDNA and a cloned portion of the genomic DNA shows that the complete cDNA sequence contains an open reading frame encoding a peptide of 662 amino acid residues in length and predicts a polypeptide with a molecular mass of 73.5 kDa, which corresponds to that detected by western blotting. Only one PEPCK gene was identified in the tomato genome. PEPCK is shown to be present in the pericarp of ripening tomato fruits by activity measurements, western blotting and mRNA analysis. PEPCK abundance and activity both increased during fruit ripening, from an undetectable amount in immature green fruit to a high amount in ripening fruit. PEPCK mRNA, protein and activity were also detected in germinating seeds and, in lower amounts, in roots and stems of tomato. The possible role of PEPCK in the pericarp of tomato fruit during ripening is discussed.     

Changes in gene expression during strawberry fruit ripening and their regulation by auxin

Changes in messenger RNA during the development of the strawberry (Fragaria ananassa Duch.), a non-climacteric fruit, were analysed by extracting total RNA and separating the in-vitro translated products by two-dimensional polyacrylamide gel electrophoresis. Alterations in numerous messenger RNAs accompanied fruit development between the immature green stage and the overripe stage, with prominent changes detected at or before the onset of ripening. A number of messenger RNAs undetectable in immature green fruit increased as the fruit matured and ripened. Others showed a marked decrease in advance of the ripening phase. A further group of messenger RNAs was prominent in immature and ripe fruit but absent just prior to the turning stage. Removing the achenes from a segment of the fruit accelerated anthocyanin accumulation in the de-achened portion and produced a pattern of translated polypeptides similar to normal ripe fruit. Application of the synthetic auxin 1-naphthaleneacetic acid to the de-achened receptacle produced a translation pattern similar to that in mature green fruit. These findings indicate that ripening in strawberry is associated with the expression of specific genes.