Why the aphid Aphis spiraecola is more abundant on clementine tree than Aphis gossypii?

Aphis spiraecola and Aphis gossypii cause harmful damages on clementine tree orchards. Weekly surveys measured the abundance of aphids (larvae, winged and wingless adults) as well as of auxiliary insects and parameters of energy metabolism. Correlatively, soluble carbohydrates, total free amino acids, free proline and condensed tannins were quantified in control and infested leaves. Both aphid species showed parallel temporal variations, but A. spiraecola was consistently more abundant regardless of the stage. Amino acids had a positive effect on both aphid species abundance, but neither condensed tannins nor auxiliary insects seemed to modulate aphid populations. Interestingly, the leaf carbohydrate content was positively correlated with the abundance of A. spiraecola, but not with that of A. gossypii. Moreover, A. gossypii's abundance was significantly down-regulated by high proline concentrations. Thus, the higher abundance of A. spiraecola could be explained by a better tolerance to high proline contents and a better conversion of foliar energy metabolites.     

Drought-Stressed Tomato Plants Trigger Bottom–Up Effects on the Invasive Tetranychus evansi

Climate change will bring more drought periods that will have an impact on the irrigation practices of some crops like tomato, from standard water regime to deficit irrigation. This will promote changes in plant metabolism and alter their interactions with biotic stressors. We have tested if mild or moderate drought-stressed tomato plants (simulating deficit irrigation) have an effect on the biological traits of the invasive tomato red spider mite, Tetranychus evansi. Our data reveal that T evansi caused more leaf damage to drought-stressed tomato plants (≥1.5 fold for both drought scenarios). Mite performance was also enhanced, as revealed by significant increases of eggs laid (≥2 fold) at 4 days post infestation (dpi), and of mobile forms (≥2 fold and 1.5 fold for moderate and mild drought, respectively) at 10 dpi. The levels of several essential amino acids (histidine, isoleucine, leucine, tyrosine, valine) and free sugars in tomato leaves were significantly induced by drought in combination with mites. The non-essential amino acid proline was also strongly induced, stimulating mite feeding and egg laying when added to tomato leaf disks at levels equivalent to that estimated on drought-infested tomato plants at 10 dpi. Tomato plant defense proteins were also affected by drought and/or mite infestation, but T. evansi was capable of circumventing their potential adverse effects. Altogether, our data indicate that significant increases of available free sugars and essential amino acids, jointly with their phagostimulant effect, created a favorable environment for a better T. evansi performance on drought-stressed tomato leaves. Thus, drought-stressed tomato plants, even at mild levels, may be more prone to T evansi outbreaks in a climate change scenario, which might negatively affect tomato production on area-wide scales.     

Comparative study of the free amino acid compositions and contents in three different botanical origins of Coptis herb

Free amino acids are important bioactive ingredients in herbs and herbal products. Coptis herbs contain a variety of free amino acids; however, studies have not yet analyzed the relationship between free amino acids and species of Coptis herbs. In the current study, the contents of 20 free amino acids in Coptis chinensis Franch., Coptis teeta Wall., and Coptis deltoidea C.Y. Cheng et Hsiao were determined using an automatic amino acid analyzer to evaluate the differences between the three species. We found that the major amino acids (Asn, Arg, and γ-aminobutyric acid (GABA)) were significantly more in C. chinensis than in the other two species. In addition, Asp content was significantly more in C. deltoidea. The study concluded that Asn can be used to identify C. deltoidea, C. chinensis, and C. teeta, and Gln and Arg can be used to distinguish C. teeta from C. chinensis and C. deltoidea. These findings suggested that free amino acids as active ingredients can be used to identify Coptis herb species.     

Distribution and biosynthesis of theanine in Theaceae plants

The theanine content of the leaves of 27 species or varieties of Theaceae plants was investigated. Theanine was present in 21 species or varieties, but in much lower amounts (<0.2 μmol/g fresh weight) than the quantity detected in Camellia sinensis var. sinensis. The major free amino acids in leaves of four species belonging to the genera Schima and Eurya, were glutamic acid, aspartic acid, glutamine, asparagine, alanine and proline and content of these amino acids is similar to or higher than theanine. Accumulation of free amino acids in these plants was generally lower than in C. sinensis var. sinensis. The biosynthetic activity of theanine, assessed by the incorporation of radioactivity from [¹⁴C]ethylamine, was detected in seedlings of two species of Schima. The theanine biosynthetic activity in roots was higher than that of leaves.     

Estimating the number of protein molecules in a plant cell: protein and amino acid homeostasis during drought

During drought stress, cellular proteostasis on the one hand and amino acid homeostasis on the other hand are severely challenged, because the decrease in photosynthesis induces massive proteolysis, leading to drastic changes in both the proteome and the free amino acid pool. Thus, we selected progressive drought stress in Arabidopsis (Arabidopsis thaliana) as a model to investigate on a quantitative level the balance between protein and free amino acid homeostasis. We analyzed the mass composition of the leaf proteome based on proteomics datasets, and estimated how many protein molecules are present in a plant cell and its subcellular compartments. In addition, we calculated stress-induced changes in the distribution of individual amino acids between the free and protein-bound pools. Under control conditions, an average Arabidopsis mesophyll cell contains about 25 billion protein molecules, of which 80% are localized in chloroplasts. Severe water deficiency leads to degradation of more than 40% of the leaf protein mass, and thus causes a drastic shift in distribution toward the free amino acid pool. Stress-induced proteolysis of just half of the 340 million RubisCO hexadecamers present in the chloroplasts of a single mesophyll cell doubles the cellular content of free amino acids. A major fraction of the amino acids released from proteins is channeled into synthesis of proline, which is a compatible osmolyte. Complete oxidation of the remaining fraction as an alternative respiratory substrate can fully compensate for the lack of photosynthesis-derived carbohydrates for several hours.

A quantitative perspective on the protein and amino acid composition of a leaf cell illustrates the dimension and specific consequences of massive proteolysis induced by severe water deficit.     

Two CAPS markers predict Verticillium wilt resistance in wild Solanum species

Verticillium wilt of potato is a persistent problem in the USA and worldwide. The disease, which is caused primarily by the fungus Verticillium dahliae, is difficult to manage, causes yield losses, and contaminates soil for subsequent plantings. Control strategies based on host resistance are seen as long-term, stable solutions, but difficult to achieve given the genetic nature of the host and the challenges associated with resistance evaluations. To provide breeders with marker-assisted selection opportunities, we generated a pair of cleaved amplified polymorphic sequence molecular markers within the coding region of Ve2, a potato gene with homology to the tomato Ve1 gene that confers resistance to V. dahliae. The position of the marker was determined according to the consensus sequences of Ve2 homologs of wild Solanum species with resistance to V. dahliae. Marker testing indicated their broad applicability, being able to track the resistance to V. dahliae in progeny containing genetic information derived from species S. chacoense, S. brevicaule, S. berthaultii, S. tarijense, and S. tuberosum. Furthermore, the two isolates of V. dahliae used in our inoculation experiments differed in virulence and demonstrated specificity for some wild potato species. Experimentation leading to the development of the markers and tests of their usefulness against a wide range of diploid potato germplasm is presented.     

Silencing of Vlaro2 for chorismate synthase revealed that the phytopathogen Verticillium longisporum induces the cross-pathway control in the xylem

The first leaky auxotrophic mutant for aromatic amino acids of the near-diploid fungal plant pathogen Verticillium longisporum (VL) has been generated. VL enters its host Brassica napus through the roots and colonizes the xylem vessels. The xylem contains little nutrients including low concentrations of amino acids. We isolated the gene Vlaro2 encoding chorismate synthase by complementation of the corresponding yeast mutant strain. Chorismate synthase produces the first branch point intermediate of aromatic amino acid biosynthesis. A novel RNA-mediated gene silencing method reduced gene expression of both isogenes by 80% and resulted in a bradytrophic mutant, which is a leaky auxotroph due to impaired expression of chorismate synthase. In contrast to the wild type, silencing resulted in increased expression of the cross-pathway regulatory gene VlcpcA (similar to cpcA/GCN4) during saprotrophic life. The mutant fungus is still able to infect the host plant B. napus and the model Arabidopsis thaliana with reduced efficiency. VlcpcA expression is increased in planta in the mutant and the wild-type fungus. We assume that xylem colonization requires induction of the cross-pathway control, presumably because the fungus has to overcome imbalanced amino acid supply in the xylem.     

VdNEP, an Elicitor from Verticillium dahliae, Induces Cotton Plant Wilting

Verticillium wilt is a vascular disease of cotton. The causal fungus, Verticillium dahliae, secretes elicitors in culture. We have generated ~1,000 5′-terminal expressed sequence tags (ESTs) from a cultured mycelium of V. dahliae. A number of ESTs were found to encode proteins harboring putative signal peptides for secretion, and their cDNAs were isolated. Heterologous expression led to the identification of a protein with elicitor activities. This protein, named V. dahliae necrosis- and ethylene-inducing protein (VdNEP), is composed of 233 amino acids and has high sequence identities with fungal necrosis- and ethylene-inducing proteins. Infiltration of the bacterially expressed His-VdNEP into Nicotiana benthamiana leaves resulted in necrotic lesion formation. In Arabidopsis thaliana, the fusion protein also triggered production of reactive oxygen species and induced the expression of PR genes. When added into suspension cultured cells of cotton (Gossypium arboreum), the fusion protein elicited the biosynthesis of gossypol and related sesquiterpene phytoalexins at low concentrations, and it induced cell death at higher concentrations. On cotton cotyledons and leaves, His-VdNEP induced dehydration and wilting, similar to symptoms caused by a crude preparation of V. dahliae elicitors. Northern blotting showed a low level of VdNEP expression in the mycelium during culture. These data suggest that VdNEP is a wilt-inducing factor and that it participates in cotton-V. dahliae interactions.     

The nitrogen content of the tomato leaf apoplast increases during infection by Cladosporium fulvum

To address the problem of the nutritional requirements of phyto-pathogenic fungi growing in planta, the environment for the intercellular biotrophic pathogen, Cladosporium fulvum Cooke, of tomato (Lycopersicon esculentum Mill.) was analysed. Using a novel technique for infiltrating the intercellular space, we measured the concentrations of 21 amino acids, nitrate and ammonia in the apoplast of the tomato leaf during infection. The concentrations of most amino acids, and total nitrogen content, increased during infection. The levels of nearly all amino acids remained relatively unchanged during an incompatible interaction. All protein amino acids were detected during infection, except cysteine and tryptophan. Most amino acids were present at a concentration between 0.1-0.7 mM. The non-protein amino acid gamma-aminobutyric acid was detected at the highest concentration (up to 2.5 mM) during the compatible interaction. Preliminary investigations on the source of the amino acids revealed that protease activity within the apoplast increased during infection and that infection induced the expression of the pathogenicity-related extracellular serine protease P69B. The nitrogen status of the infecting fungus and sources for the additional amino acids are discussed.     

Maintenance of Sex-Related Genes and the Co-Occurrence of Both Mating Types in Verticillium dahliae

Verticillium dahliae is a cosmopolitan, soilborne fungus that causes a significant wilt disease on a wide variety of plant hosts including economically important crops, ornamentals, and timber species. Clonal expansion through asexual reproduction plays a vital role in recurring plant epidemics caused by this pathogen. The recent discovery of recombination between clonal lineages and preliminary investigations of the meiotic gene inventory of V. dahliae suggest that cryptic sex appears to be rare in this species. Here we expanded on previous findings on the sexual nature of V. dahliae. Only 1% of isolates in a global collection of 1120 phytopathogenic V. dahliae isolates contained the MAT1-1 idiomorph, whereas 99% contained MAT1-2. Nine unique multilocus microsatellite types comprised isolates of both mating types, eight of which were collected from the same substrate at the same time. Orthologs of 88 previously characterized sex-related genes from fungal model systems in the Ascoymycota were identified in the genome of V. dahliae, out of 93 genes investigated. Results of RT-PCR experiments using both mating types revealed that 10 arbitrarily chosen sex-related genes, including MAT1-1-1 and MAT1-2-1, were constitutively expressed in V. dahliae cultures grown under laboratory conditions. Ratios of non-synonymous (amino-acid altering) to synonymous (silent) substitutions in V. dahliae MAT1-1-1 and MAT1-2-1 sequences were indistinguishable from the ratios observed in the MAT genes of sexual fungi in the Pezizomycotina. Patterns consistent with strong purifying selection were also observed in 18 other arbitrarily chosen V. dahliae sex-related genes, relative to the patterns in orthologs from fungi with known sexual stages. This study builds upon recent findings from other laboratories and mounts further evidence for an ancestral or cryptic sexual stage in V. dahliae.     

Island Cotton Gbve1 Gene Encoding A Receptor-Like Protein Confers Resistance to Both Defoliating and Non-Defoliating Isolates of Verticillium dahliae

Verticillium wilt caused by soilborne fungus Verticillium dahliae could significantly reduce cotton yield. Here, we cloned a tomato Ve homologous gene, Gbve1, from an island cotton cultivar that is resistant to Verticillium wilt. We found that the Gbve1 gene was induced by V. dahliae and by phytohormones salicylic acid, jasmonic acid, and ethylene, but not by abscisic acid. The induction of Gbve1 in resistant cotton was quicker and stronger than in Verticillium-susceptible upland cotton following V. dahliae inoculation. Gbve1 promoter-driving GUS activity was found exclusively in the vascular bundles of roots and stems of transgenic Arabidopsis. Virus-induced silencing of endogenous genes in resistant cotton via targeting a fragment of the Gbve1 gene compromised cotton resistance to V. dahliae. Furthermore, we transformed the Gbve1 gene into Arabidopsis and upland cotton through Agrobacterium-mediated transformation. Overexpression of the Gbve1 gene endowed transgenic Arabidopsis and upland cotton with resistance to high aggressive defoliating and non-defoliating isolates of V. dahliae. And HR-mimic cell death was observed in the transgenic Arabidopsis. Our results demonstrate that the Gbve1 gene is responsible for resistance to V. dahliae in island cotton and can be used for breeding cotton varieties that are resistant to Verticillium wilt.     

Morphological and molecular characterization of the strawberry black leaf spot pathogen referred to as the strawberry pathotype of Alternaria alternata

The remaining unclarified taxon among the seven known pathotypes of host-selective toxin (HST)-producing Alternaria alternata, namely, the strawberry pathotype (the strawberry black leaf spot pathogen), is taxonomically revised and re-described herein. According to our morphological observations, reference isolates of strawberry and Japanese pear pathotypes, which are toxic to leaves of Japanese pear ‘Nijisseiki’, have conidia that are formed in chains of 3–13, usually without lateral branches, after 7?d incubation on potato-carrot agar. The mean size of the conidia is 27–31?×?11–13?μm. Morphological characteristics of the examined isolates are identical to those of A. gaisen rather than A. alternata. A phylogenetic tree obtained by analysis of a combined dataset of ITS, gapdh, rpb2, tef1, Alt a 1, and endoPG sequences also strongly supports both pathotypes as one species, A. gaisen. We re-describe the fungus as A. gaisen Nagano ex Bokura and propose two formae speciales of the species, A. gaisen f. sp. fragariae producing AF-toxin and f. sp. pyri producing AK-toxin. The epitype specimen and ex-epitype culture of A. gaisen are newly designated.     

Properties of capsaicinoids for the control of fungi and oomycetes pathogenic to pepper

Capsaicinoids are pungent compounds found in pepper (Capsicum spp.) fruits. Capsaicin showed antimicrobial activity in plate assays against seven isolates of five species of fungi and nine isolates of two species of oomycetes. The general trend was that oomycetes were more inhibited than fungi. Assays of capsaicin biosynthetic precursors suggest that the lateral chain of capsaicinoids has more inhibitory activity than the phenolic part. In planta tests of capsaicinoids (capsaicin and N‐vanillylnonanamide) applied to the roots demonstrated that these compounds conferred protection against the pathogenic fungus Verticillium dahliae and induced both chitinase activity and expression of several defence‐related genes, such as CASC1, CACHI2 and CABGLU. N‐Vanillylnonanamide infiltrated into cotyledons confers systemic protection to the upper leaves of pepper against the fungal pathogen Botrytis cinerea. In wild‐type tomato plants such cotyledon infiltration has no protective effect, but is effective in the Never‐ripe tomato mutant impaired in ethylene response. A similar effect was observed in tomato after salicylic acid infiltration.     

Salivary amino acids in Lygus species (Heteroptera:Miridae)

Free and protein-bound amino acids were investigated in the phytophagous bug Lygus rugulipennis and its salivary gland. Over 38 substances were separated. The total content of amino compounds in the insects was about 1400 μmol/g fr. wt (16% by weight), of which 97% was amino acid residues in proteins.The salivary glands, which comprise about 1.5% of the live weight of the insects, contain 3.5% of the total free amino acids and 1% of the whote insect. Free and protein-bound amino acids comprise, respectively, about 1.4 and 11.6% of the fresh weight of the gland. The total concentration of free amino acids in the saliva was estimated to range from 0.5 to 2.2% by weight (ca. 0.1 M).The composition of free amino acids in the salivary gland of Lugus varies markedly. In four studied species (L. rugulipennis, L. gemellatus, L. pratensis, L. punctatus), the most abundant compounds were proline, arginine, lysine, leucine, glutamic acid, methionine sulphoxide and glycerophosphoethanolamine. In whole specimens of L. rugulipennis the predominant free amino acids were proline, alanine, taurine, glutamic acid, glutamine and methionine sulphoxide. The most abundant amino acids in proteins were glutamic and aspartic acid, glycine, alanine and leucine. The results indicate that the amino acid composition in the salivary glands of Lygus species does not differ markedly from that of the whole insect. The functions of salivary amino acids are discussed.     

Non-protein amino acids of Acacia species and their effect on the feeding of the acridids Anacridium melanorhodon and Locusta migratoria

The leaves of Acacia species have been found to contain homoarginine, pipecolic acid and 4-hydroxy-pipecolic acid. The nymphs of the tree locust Anacridium melanorhodon, which feed on the leaves of Acacia species, were not inhibited from feeding on palatable media containing concentrations of these amino acids equivalent to, or greater than, those found in the leaves. The graminivorous Locusta migratoria was more sensitive to these compounds, inhibitory effects being observed at concentrations comparable to those found in the leaves. The inhibitory effects of mixtures of homoarginine and pipecolic acid were additive in A. melanorhodon but not in L. migratoria. Three of the non-protein amino acids found in the seeds of Acacia species, 2,3-diaminopropionic acid, 2-amino-3-acetylaminopropionic acid and 2-amino-3-oxalylaminopropionic acid, were more effective inhibitors of feeding in Anacridium than were the leaf amino acids.     

Enzymology of Glutamine Metabolism Related to Senescence and Seed Development in the Pea (Pisum sativum L.)

The metabolism of glutamine in the leaf and subtended fruit of the aging pea (Pisum sativum L. cv. Burpeeana) has been studied in relation to changes in the protein, chlorophyll, and free amino acid content of each organ during ontogenesis. Glutamine synthetase [EC 6.3.1.2] activity was measured during development and senescence in each organ. Glutamate synthetase [EC 2.6.1.53] activity was followed in the pod and cotyledon during development and maturation. Maximal glutamine synthetase activity and free amino acid accumulation occurred together in the young leaf. Glutamine synthetase (in vitro) in leaf extracts greatly exceeded the requirement (in vivo) for reduced N in the organ. Glutamine synthetase activity, although declining in the senescing leaf, was sufficient (in vitro) to produce glutamine from all of the N released during protein hydrolysis (in vivo). Maximal glutamine synthetase activity in the pod was recorded 6 days after the peak accumulation of the free amino acids in this organ.

In the young pod, free amino acids accumulated as glutamate synthetase activity increased. Maximal pod glutamate synthetase activity occurred simultaneously with maximal leaf glutamine synthetase activity, but 6 days prior to the corresponding maximum of glutamine synthetase in the pod. Cotyledonary glutamate synthetase activity increased during the assimilatory phase of embryo growth which coincided with the loss of protein and free amino acids from the leaf and pod; maximal activity was recorded simultaneously with maximal pod glutamine synthetase.

We suggest that the activity of glutamine synthetase in the supply organs (leaf, pod) furnishes the translocated amide necessary for the N nutrition of the cotyledon. The subsequent activity of glutamate synthetase could provide a mechanism for the transfer of imported amide N to alpha amino N subsequently used in protein synthesis. In vitro measurements of enzyme activity indicate there was sufficient catalytic potential in vivo to accomplish these proposed roles.

     

Interactions Among Pratylenchus penetrans,P. scribneri,and Verticillium dahliae in the Potato Early Dying Disease Complex

Microplots were infested with combinations of the fungus Verticillium dahliae and Pratylenchus penetrans and P. scribneri to test for individual and combined effects of these organisms on potato yield and nematode reproduction. Verticillium dahliae alone caused yield losses in all 3 years of the experiment, and the interaction between P. penetrans and V. dahliae was significant (P ≤ 0.05) in 2 years. Pratylenchus penetrans alone caused yield losses in 2 years and P. scribneri alone caused yield losses in 1 year. No two-way or three-way interaction was found involving P. scribneri. In 1987, reproduction for low densities of P. penetrans was 5 times higher when P. scribneri was also present than when it was absent, and 3.5 times higher in 1988. In nematode species mixtures, reproduction of P. scribneri was decreased by V. dahliae in 1987-88. The final population density of P. scribneri was negatively affected by V. dahliae and positively related to the initial proportion of P. scribneri to P. penetrans. In species mixtures with proportions of P. penetrans ranging from 0.1 to 0.5, reproduction of P. penetrans was negatively affected by V. dahliae and decreased linearly in relation to the increase in the initial proportion of P. penetrans in both years. The final population density of P. penetrans was affected only by V. dahliae.