Influence of Colletotrichum simmondsii R. G. Shives & Y. P. Tan infection on selected primary and secondary metabolites in strawberry (Fragaria x ananassa Duch.) fruit and runners

The effect of Colletotrichum simmondsii infection on the contents of sugars, organic acids, and individual phenolic compounds was investigated in strawberry cultivar ‘Clery’. Primary metabolites were determined with the use of HPLC and secondary metabolites further confirmed with HPLC-MS. Colletotrichum simmondsii caused a decrease in sucrose and an increase in fructose and glucose in strawberry fruit. A significant decrease in the content of malic and citric acids was recorded in infected fruit. 12 forms of ellagic acid, nine flavanols and eight flavonols were identified in strawberry runners and nine forms of ellagic acid, six flavanols, seven flavonols and four anthocyanins in strawberry fruit. Significant differences in individual phenolic compounds in strawberry fruit were detected at the beginning of the infection compared to non-infected fruit. Specifically, ellagic acids significantly increased, flavonols generally decreased, and flavanols and anthocyanins increased with the progression of infection. Similarly, some forms of ellagic acid increased and others decreased in infected runners, procyanidins generally decreased and flavonols, increased but the differences were much less prominent.     

Phenolic compounds as defence response of pepper fruits to Colletotrichum coccodes

Qualitative and quantitative changes of individual and total phenolics induced by Colletotrichum coccodes fungal infection have been studied in two susceptible sweet pepper cultivars ‘Soroksari’ and ‘Bagoly’, and the role of soluble phenolic compounds in plant's defence mechanism has been evaluated. Three distinct parts were analysed on pepper fruit: healthy tissue, anthracnose lesion, and bordering tissue, and individual phenolic compounds have been identified with the use of HPLC-MS system. In pepper fruit pericarp 21 phenolic compounds have been determined; the prevalent apigenin, quercetin and luteolin glycosides, chlorogenic acid and one chrysoeriol glucoside. C. coccodes infection increased the accumulation of chlorogenic acid, chrysoeriol glucoside, quercetin and luteolin glycosides in infected bordering tissue of both analysed pepper cultivars compared to healthy pepper tissue or symptomatic spot. Total apigenin derivatives did not show a significant increase in bordering tissue compared to the healthy pepper fruit in contrast to other groups of phenolics. This suggests a lesser role of apigenin glycosides in pepper plant defence against the Colletotrichum fungus. Intense phenolic synthesis was characteristic for the bordering zone between the healthy and infected plant tissue resulting in higher total phenolic content which might hinder the fungus to spread from the infected cells into the healthy tissue.     

The development of tea blister caused by Exobasidium vexans in tea (Camellia sinensis) correlates with the reduced accumulation of some antimicrobial metabolites and the defence signals salicylic and jasmonic acids

Blister blight (causal agent, Exobasidium vexans) is an economically devastating disease of tea (Camellia sinensis). To determine what metabolite changes occur with tea blister that could be linked to disease progression, metabolomic approaches were used on E. vexans infected tea from a Darjeeling (India) plantation. Samples were classified according to disease phenotypes, i.e. either healthy or at one of three stages of disease progression. Initial metabolite fingerprinting using Fourier transform infrared (FTIR) spectroscopy indicated that metabolite changes could be related to disease stage. Electrospray ionization mass spectrometry (ESI‐MS) highlighted caffeine and flavonoid metabolism changes as disease progressed. High‐performance liquid chromatography (HPLC) with online photodiode array detection and electrospray ionization‐tandem mass spectrometry (HPLC‐PDA‐ESI/MSⁿ) was used to characterize the caffeine, flavan‐3‐ol, flavone and flavonol profiles. There were increases in quercetin and kaempferol glucosides, kaempferol triglycosides and some catechin‐class antioxidants, but also substantial reductions in apigenin and myricetin glycosides and, particularly, caffeine as disease progressed. The content of important defence hormones, salicylic acid and jasmonic acid, was also reduced in blister blight diseased samples. Thus, E. vexans infections perturb defence signalling and reduce many potentially antimicrobial compounds, such as caffeine, to aid disease progression.     

Coffee resistance to Colletotrichum kahawae is associated with lignification, accumulation of phenols and cell death at infection sites

Histological and ultrastructural studies were undertaken to compare Colletotrichum kahawae growth and the sequence of responses it induced in resistant and susceptible coffee genotypes. Coffee resistance was characterized by a restricted fungal growth associated with hypersensitive-like cell death and early accumulation of phenolic compounds, such as flavonoids (cytoplasmic contents) and hydroxycinnamic acid derivatives (cell walls). This accumulation of phenols in the cell walls preceded their lignification and thickening. In the susceptible genotype, a late accumulation of hydroxycinnamic acid derivatives in a number of cell walls and the encasement of some intracellular hyphae were also observed, but these delayed host responses did not prevent fungal growth and sporulation.     

In vitro and in planta nematicidal activity of Fumaria parviflora (Fumariaceae) against the southern root‐knot nematode Meloidogyne incognita

Root and stem extracts of Fumaria parviflora showed strong nematicidal activity against Meloidogyne incognita in in vitro and in planta experiments. Phytochemical screening of F. parviflora revealed the presence of seven classes of bioactive compounds (alkaloids, flavonoids, glycosides, tannins, saponins, steroids and phenols). Quantitative determination of the plant extracts showed the highest percentages of alkaloids (0·9 ± 0·04) and saponins (1·3 ± 0·07) in the roots and total phenolic contents in the stem (16·75 ± 0·07 μg dry g^?1). The n‐hexane, chloroform, ethyl acetate and methanol extracts of roots and stems at concentrations of 3·12, 6·24, 12·5, 25·0 and 50·0 mg mL^?1, significantly inhibited hatching and increased mortality of second‐stage juveniles (J2s) compared with water controls. Percentage J2 mortality and hatch inhibition were directly related to exposure time. In pot trials with tomato cv. Rio Grande, root and stem extracts at concentrations of 1000, 2000 and 3000 ppm, applied as soil drenches, significantly reduced the number of galls, galling index, eggs masses, eggs and reproduction factor compared with the water control. Regardless of concentration, all the extracts significantly increased the host plant growth parameters studied. The n‐hexane extracts from the roots and stem were the most active, followed by the methanol ones, at all concentrations. The in vitro and in planta results suggest that extracts from the roots and stem of F. parviflora may be potential novel nematicides.     

Current situation and characterization of Pseudomonas syringae pv. actinidiae on kiwifruit in Galicia (northwest Spain)

Bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae (Psa), is a disease that is spreading rapidly in several kiwifruit‐producing countries, causing significant economic losses. In 2011, it was detected for the first time in Spain, in the south of Galicia (northwest Spain). Kiwifruit orchards were therefore inspected and sampled in 2011 and 2012 to determine the pathogen distribution, and the isolates obtained were characterized by morphology, fatty acids profile, biochemical tests and molecular techniques. Isolates were obtained from Actinidia deliciosa ‘Hayward’ (from leaves, canes, flower buds, fruits and roots), from A. deliciosa ‘Summer’, from Actinidia chinensis ‘Jin Tao’ (from canes and leaves) and from A. chinensis pollinator ‘Belén’ (from canes). Results of the analysis of the cfl gene (phytotoxin production‐related), the tox–argK gene cluster and phylogenetic analysis of the cts gene demonstrated that all Psa isolates from northwest Spain correspond to the Psa3 population, which includes strains of haplotype 2. This is the first record of Psa3 and haplotype 2 in Spain.     

The response of phenolic compounds in grapes of the variety ‘Chardonnay’ (Vitis vinifera L.) to the infection by phytoplasma Bois noir

The study was carried out on phytoplasma susceptible grapevine variety ‘Chardonnay’ (Vitis vinifera L.). The changes in total and individual phenolics, with a focus on hydroxycinnamic acids, flavanols and flavonol contents, were studied in phytoplasma-symptomatic and non-symptomatic berries of Bois noir (BN) infected and uninfected vines. Evident responses to BN infection at veraison have been monitored in a decreased accumulation of caftaric and coutaric acids, p-coumaroyl hexose, procyanidin B1, procyanidin trimer as well as of quercetin-3-O-glucoside, quercetin-3-O-glucuronide and quercetin-3-O-xyloside. At berry softening BN infection statistically increased the content of total phenolics, hydroxycinnamic acids and flavanols, but decreased the flavonol contents, especially at phytoplasma-symptomatic berry skins. Later, at harvest, the BN infection caused an additional significant decrease of coutaric acid and p-coumaroyl pentose contents, moreover of procyanidin B1 and procyanidin dimmers (1, 2, and 3), trimer, kaempferol-3-O-glucoside and of most identified quercetins, except of quercetin-3-O-xyloside. At harvest, non-symptomatic berries from infected plants showed similar dynamics in the total phenolic content compared to berry skins from uninfected plants, but in total flavanols and flavonols content similarity to those symptomatic was observed. The latter decreases grape quality and its antioxidant potential. The Bois noir disease showed specific, local and growth-phase-induced responses regarding the content of phenolics in berry skins, where in particular the differences between phytoplasma-symptomatic and non-symptomatic grapes have to be underlined.     

Colletotrichum acutatum interactions with unripe and ripe strawberry fruits and differential responses at histological and transcriptional levels

Microscopic investigations were conducted into the interaction of Colletotrichum acutatum on white and red strawberry (Fragaria ×ananassa) fruit surfaces. The results showed that, whilst the early interaction events were similar in both white and red fruits, after 24 h fungal colonization dramatically varied: in white fruits C. acutatum became quiescent as melanized appressoria, but on red fruits it displayed subcuticular necrotrophic invasion. A microarray analysis of white and red strawberries after 24 h of interaction with C. acutatum was performed, in order to reveal differences in gene expression possibly related to the different susceptibility of unripe and ripe fruits. Epi/catechin‐related genes and fatty acid metabolism genes, involved in the production of quiescence‐related molecules such as flavan‐3‐ols, proanthocyanidins and antifungal dienes, were found to be regulated during strawberry ripening, supporting a role for these molecules as preformed defence mechanisms. Besides several genes commonly regulated upon pathogen interaction, different genes were specifically transcribed only in white or red challenged fruits; a number of these, such as those coding for lectin and polyphenol oxidase, possibly account for specific pathogen‐induced responses. The putative biological role of these genes in the different susceptibility of fruits to C. acutatum is discussed.     

Biochemical response of grapevine variety ‘Chardonnay’ (Vitis vinifera L.) to infection with grapevine yellows (Bois noir)

This study was carried out on the leaves of phytoplasma susceptible grapevine variety ??Chardonnay?? (Vitis vinifera L.), and included research of the alterations in chlorophyll and carotenoid contents, contents of phenol compounds and in related enzymes activity in the phenylpropanoid pathway during the Bois noir (BN) infection. Phytoplasma-infected leaves showed reduced contents of chlorophylls and carotenoids, which promoted their susceptibility to oxidative reactions. Furthermore, modulation of flavonoid biosynthesis occurred in infected leaves, leading to an increased activity of phenylalanine ammonia lyase, chalcone synthase/chalcone isomerase, flavanone 3-hydroxylase and polyphenoloxidase, but to a decreased peroxidase activity. Phytoplasma infection led to an increase of the contents of hydroxycinnamic acids (caftaric acid, sinapic acid glucose derivate and coutaric acid), flavanols (procyanidin B1, procyanidin dimer 3, catechin, epicatechin) and flavonols (quercetin 3-O-glucuronide, quercetin 3-O-glucoside) especially in the period up to vérasion. The study demonstrated that at certain phenological key-stages infection with phytoplasma (BN) induced different alterations in enzyme activities and in the contents of biochemical compounds from primary and secondary metabolism.     

Examination of latent infection in raspberry canes with Phytophthora rubi and P. idaei and transmission in micropropagation

Buds of raspberry canes from naturally and artificially infected root stocks were tested by nested PCR for the latent presence of Phytophthora rubi and P. idaei to establish the risk of introducing these pathogens into micropropagation. Fifteen out of 201 buds tested positive for P. rubi and only 1 out of 176 for P. idaei. These infections were not restricted to buds in the lower part of the cane and mostly do not appear to spread systemically up from the rootstock but seem to be caused by secondary infections of the canes. Buds from symptomless canes from infected root stocks were used for micropropagation and P. rubi could be detected in 5.8% of the obtained microplants after ten generations in one of the two trials. This indicates that micropropagation procedures are not absolutely infallible in eliminating infection and underlines the necessity of a zero tolerance towards diseases in the starting material.     

Occurrence and distribution of Raspberry bushy dwarf virus in commercial Rubus plantations in England and Wales

Serological surveys for Raspberry bushy dwarf virus (RBDV) made between 1995 and 1997 and covering ≈ 10% of the commercial farms growing Rubus (red raspberry and hybrid berries) in England and Wales showed that this virus was present on approximately one-quarter of all farms and in approximately one-sixth of all plots tested. RBDV was found in all of the four main raspberry cultivars being grown at that time (Autumn Bliss, Glen Moy, Glen Prosen and Leo), in Loganberry and in Tayberry. Fifteen RBDV genotypes (including two that appeared to be mixed) were identified using RT-PCR/RFLPs, but the majority of genotypes were found only rarely. Of the RBDV isolates tested, two genotypes each comprised 12·5% and another 46·4%. None of the three most common genotypes was associated solely with single Rubus cultivars and vice versa . It is suggested that two separate outbreaks of RBDV are occurring in England and Wales. One outbreak comprises the most frequent genotype combined with one of the moderately frequent genotypes; this outbreak is largely confined to the main growing areas and is either spreading between farms or coming from multiple local sources. Circumstantial evidence suggests that these isolates (and hence this first outbreak) are of the RB pathotype. The second outbreak consists of the other moderately frequent genotype and those genotypes which are less common. These genotypes appear to be more scattered across England and Wales and seem more likely to be coming from local sources and not to be spreading naturally between commercial farms.     

Differential induction of redox sensitive extracellular phenolic amides in potato

This study focuses on the differential induction of extracellular phenolic amides that accumulate in potato cell suspensions during the first few hours of the interaction between these plant cells and either bacterial pathogens or pathogen-related elicitors. Using suspension cells of Solanum tuberosum we identified 4 hydroxycinnamic acid amides that accumulate in the extracellular environment. Treatment of the suspension cells with pathovars of the plant pathogens Pseudomonas syringae or Ralstonia solanacearum or with pathogen-related elicitors changed the composition of the extracellular phenolic amides within hours and the composition differed for each treatment. Some of the phenolic amides were sensitive to oxidative stress; when suspension cells were treated with bacterial strains or elicitors that triggered an oxidative burst, the phenolics were oxidized and depleted for the duration of the burst. Other critical parameters that affected the qualitative and quantitative makeup of these phenolic amides were plant cell age and density.     

Effects of three esca-associated fungi on Vitis vinifera L.: II. Characterization of biomolecules in xylem sap and leaves of healthy and diseased vines

Secondary metabolites and host defense compounds were shown to occur in xylem sap, and leaves of Vitis vinifera cv. Italia and cv. Matilde naturally infected by the esca-associated fungi Phaeomoniella chlamydospora (Pch), Togninia minima (Tmi) and Fomitiporia mediterranea (Fme). Samples of xylem sap and leaves were collected from healthy vines and from vines showing severe symptoms of brown wood-streaking caused by Pch and Tmi, or from vines with symptoms of both brown wood-streaking and white rot caused by Fme. Xylem sap collection was carried out during the early spring of 2003 and 2004, corresponding to the phenological phases: (A) cotton bud; (B) green tip; (C) leaves out; (D) stretched out leaves; and (E) visible clusters. In the present work we have studied the accumulation of biomolecules (pentaketides and α-glucans), host defense compounds (benzaldehydes, benzoic acid and cinnamic acid derivatives, flavonols, flavanols, flavan-3-ol derivatives and stilbenes) at different stages of grapevine development. Accumulation and changes in total phenolics and recurring phenolics, and of three phytotoxic secondary metabolites (scytalone, isosclerone and pullulan) were analyzed by HPLC. On comparing results for cv. Italia and cv. Matilde, it can be seen that phenolic concentrations are strongly related to the cv.     

Wheat leaf resistance to Pyrenophora tritici‐repentis induced by silicon activation of phenylpropanoid metabolism

Tan spot caused by Pyrenophora tritici‐repentis is a disease present in all wheat‐producing countries and silicon (Si) treatment of wheat plants has been shown to increase plant resistance to tan spot. In this study, the effect of phenylpropanoid metabolism on resistance to tan spot was evaluated and some phenolic compounds that accumulated in response to P. tritici‐repentis attack were identified. Furthermore, the effect of Si on phenylalanine ammonia‐lyase (PAL) activity and phenolic compound accumulation were determined in situ. Antifungal activity of differentially accumulated phenolic compounds was also evaluated in in vitro tests. Results showed that the increase in concentration of phenolic compounds was greatest at the onset of infection, and that some compounds showed fungitoxic effects including fungal tip swelling, granulation of germ tube and hyphae, and hyphal hyperbranching. Silicon‐induced reduction in both lesion size and tan spot disease progression were associated with activation of phenylpropanoid metabolism. PAL activity and accumulation of antifungal phenolic compounds were greater in pathogen‐inoculated plants supplied with Si. In these plants, fluorescence indicative of accumulation of phenolic compounds occurred early in epidermal cells and its intensity increased during the evaluation period, showing higher numbers of fluorescent cells around infected cells. Thus, the combined responses of cell fluorescence at sites of infection, increased PAL activity and accumulation of phenols indicate that Si strengthened wheat defence responses to infection by P. tritici‐repentis, reducing the severity of tan spot.     

Benzo-(1,2,3)-thiadiazole-7-carbothioic S-methyl ester (BTH) stimulates defense reactions in <Emphasis Type="Italic">Xanthosoma sagittifolium</Emphasis>

The root rot disease caused by Pythium myriotylum is responsible for about 70% of cocoyam production loss in Cameroon. The potential of benzo-(1,2,3)-thiadiazole-7-carbothioic S-methyl ester (BTH) to trigger resistance in cocoyam (Xanthosoma sagittifolium) plants against P. myriotylum was investigated. Under controlled conditions, BTH was an efficient elicitor of some defense reactions in cocoyam. Application of 0.2 mg ml⁻¹ of BTH on leaves 7 days before inoculation of roots with P. myriotylum enhanced the activities of peroxidase (Pox) and polyphenoloxidase (PPO) as well as the total phenolic content. This resistance was noted as a decrease in disease incidence and severity in BTH-treated plants. This increase in Pox activities was correlated with two new isoforms in a white (sensitive) cultivar inoculated after stimulation. In a yellow (resistant) cultivar, stimulation was characterized by the appearance of one isoform. Qualitative analysis of phenolic compounds by HPLC showed an increase of hydroxycinnamic and flavonoid derivatives after inoculation. We also observed the appearance of a new caffeoylshikimic acid derivative after stimulation followed by inoculation of both cultivars. The findings indicated that the pattern of induction is different and depends on the variety.     

Antifungal capacity of major phenolic compounds of Olea europaea L. against Phytophthora megasperma Drechsler and Cylindrocarpon destructans (Zinssm.) Scholten

The phenolic composition of olive roots and stems was studied by high performance liquid chromatography–mass spectrometry. The in vivo levels of the principal phenolic compounds found in olive plants infected by Phytophthora megasperma Drechsler and Cylindrocarpon destructans (Zinssm.) Scholten differed from the levels observed in non-infected plants. When the antifungal activity of these compounds against both fungi was studied in vitro, the most active were quercetin and luteolin aglycons, followed by rutin, oleuropein, p-coumaric acid, luteolin-7-glucoside, tyrosol and catechin. Microscopic study showed that these phenolic compounds affected the growth, morphology and ultrastructure of the fungi. Taken together, these findings suggest that the phenolic compounds present in olive plants play an active role in the protection against pathogen attack.     

Reduction of clubroot (Plasmodiophora brassicae) formation in Arabidopsis thaliana after treatment with prohexadione‐calcium,an inhibitor of oxoglutaric acid‐dependent dioxygenases

Recently, flavonoids were shown to modulate the outcome of clubroot development in Arabidopsis thaliana after infection with the obligate biotrophic pathogen Plasmodiophora brassicae. Therefore, the development of clubroot disease was investigated in Arabidopsis after treatment with prohexadione‐calcium (ProCa), an inhibitor of ascorbic acid/2‐oxoglutaric acid‐dependent dioxygenases such as flavanone‐3‐hydroxylase. The treatment resulted in a reduction of the flavonols quercetin and kaempferol in clubroots, whereas the precursor naringenin highly accumulated. The root system of ProCa‐treated plants was better developed although galls were still visible. Thus, ProCa treatment resulted in reduced gall size. Flavonoids are thought to inhibit polar auxin transport by modulating auxin efflux carriers. It was investigated whether the auxin response might change as a consequence of the accumulation of naringenin in ProCa‐treated plants. In the areas of gall development an auxin response was indicated by the auxin‐responsive promoter DR5 coupled to the reporter β‐glucuronidase (GUS), whereas very little staining was found in healthy root parts. No differences in GUS activity were found between P. brassicae‐infected and ProCa‐treated plants, and plants only infected with P. brassicae, indicating that the effect of ProCa treatment on clubroot reduction is not via changes in auxin responses. As ProCa is also an inhibitor of late steps in gibberellin biosynthesis, a specific gibberellin biosynthesis inhibitor, chlormequatchloride (CCC), was tested on club development. However, CCC did not reduce disease symptoms, indicating that the observed reduced gall development was not because of gibberellin biosynthesis inhibition by ProCa.     

Metabolite accumulation in strawberry (Fragaria × ananassa Duch.) fruits and runners in response to Colletotrichum nymphaeae infection

The composition of non-infected and Colletotrichum nymphaeae infected strawberry fruits (cultivar ‘Asia') in three different stages of ripeness were evaluated. Additionally, the effect of infection on strawberry runners was monitored. C. nymphaeae infection caused a significant increase in total sugar content in mature fruits (1.1 fold) and a decrease in total sugar content in semi-mature fruits (1.4 fold) and organic acid content in all stages of ripeness (1.7 fold). Ellagic acid derivatives (1.9 fold), flavanols (1.5 fold) and flavonols (5.1 fold) significantly increased during all stages of ripeness after infection. The pattern of phenolic accumulation in strawberry runners was altered by C. nymphaeae infection in contrast to strawberry fruits as most of identified phenols decreased as a response to the infection.     

Co‐infection by Botryosphaeriaceae and Ilyonectria spp. fungi during propagation causes decline of young grafted grapevines

Decline of newly planted, grafted grapevines is a serious viticultural problem worldwide. In the Riverina (New South Wales, Australia), characteristic symptoms include low fruit yields, very short shoots and severely stunted roots with black, sunken, necrotic lesions. To determine the cause, roots and wood tissue from affected plants in 20 vineyards (Vitis vinifera cv. Chardonnay grafted to V. champini cv. Ramsey rootstock) were assayed for microbial pathogens. Ilyonectria spp. (I. macrodidyma or I. liriodendra, producers of phytotoxin brefeldin A, BFA, and cause of black foot disease of grapevines) and Botryosphaeriaceae spp. (predominantly Diplodia seriata) were isolated from rootstocks of 100 and 95% of the plants, respectively. Togninia minima and Phaeomoniella chlamydospora (cause of grapevine Petri disease) were isolated from 13 and 7% of affected plants, respectively. All Ramsey rootstock stems of grafted plants sampled from a supplier nursery were infected with Ilyonectria spp. and D. seriata. Diplodia seriata, but not Ilyonectria spp., was also isolated from 25% of canes sampled from the rootstock source block. Root inoculation of potted, disease‐free Chardonnay plants with Ilyonectria isolates from diseased vineyards caused typical disease symptoms, while co‐inoculation with Botryosphaeriaceae spp. increased disease severity. This is the first study to show that a major cause of young grapevine decline can be sequential infection by Botryosphaeriaceae from rootstock cuttings and Ilyonectria spp. from nursery soil. Although the Petri disease fungi were less common in young declining grafted grapevines in the Riverina, they are likely to contribute to the decline of surviving plants as they mature.