Effects of three esca-associated fungi on Vitis vinifera L.: I. Characterization of secondary metabolites in culture media and host responses to the pathogens in calli

Phaeomoniella chlamydospora (Pch) and Togninia minima (Tmi) produced scytalone, isosclerone and pullulans in liquid cultures, as well as in calli. Secondary metabolites and host defense compounds were shown to occur in calli of Vitis vinifera cv. Italia and cv. Matilde infected by the esca-associated fungi Pch, Tmi and Fomitiporia mediterranea (Fme). Calli of both cvs. were grown as dual cultures with Pch, Tmi and Fme. The fungi grew well in the presence of calli of both cvs., but callus growth was reduced. Accumulation and changes of total phenolics and recurring phenolics, and of two phytotoxic pentaketides and pullulans were analyzed by HPLC. On comparing results for cv. Italia and cv. Matilde, it can be seen that concentrations of phenolics are strongly related to the cv. The paper discusses the possible relationship between melanin biosynthesis in Pch and Tmi, which utilize pentaketide metabolites as intermediates and their pathogenicity related to phytotoxity of scytalone and isosclerone.     

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.     

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.     

Effects of three esca-associated fungi on Vitis vinifera L.: III. Enzymes produced by the pathogens and their role in fungus-to-plant or in fungus-to-fungus interactions

When the esca-associated fungi Phaeomoniella chlamydospora (Pch), Togninia minima (Tmi) and Fomitiporia mediterranea (Fme) were grown in liquid stationary cultures, it was seen that they were able to live in media containing resveratrol (RES) or tannic acid (TA) as the sole carbon source and that the fungi were able to convert both compounds. Particular attention is paid here to detecting RES and TA conversion. Pch, Tmi and Fme were partially inhibited by RES or TA. Pch, Tmi and Fme produced extracellular tannase, laccase and peroxidase enzymes in liquid or agarized cultures, whether glucose was present or not. When colonies of Pch, Tmi and Fme were confronted, they showed spatially and temporally heterogeneous patterns of laccase and peroxidase activity. The results indicate the non-synergistic, competitive association of Pch and Tmi and the inhibition of Fme growth. Muconic acid, a well-known intermediate in a large number of lignin and phenol oxidative processes, can partly or completely inhibit the lignolytic agent Fme, but is tolerated by Pch and Tmi. An explanation for wood pigmentation patterns by Pch, Tmi and Fme is given.     

Effects of three esca-associated fungi on Vitis vinifera L.: IV. Diffusion through the xylem of metabolites produced by two tracheiphilous fungi in the woody tissue of grapevine leads to esca-like symptoms on leaves and berries

Two naphthalenone pentaketides (scytalone and isosclerone) and α-glucans (pullulan) are produced in vitro and in planta by Phaeomoniella chlamydospora (Pch) and Togninia minima (Tmi), two tracheiphilous fungi associated with the ‘esca’ disease of grapevines. The possible role of such fungal metabolites in inducing symptoms on leaves and berries was studied in a vineyard of Vitis vinifera cv. Italia located in southern Italy. During early spring, pruning off two to four branches per vine allowed samples of xylem sap to be collected. Vine bleeding, assessed as ml day⁻¹ vine⁻¹, reached its maximum at bud burst and stopped within 28 days. The total amount of sap collected from healthy vines was about a quarter of that from esca-affected vines. During the same period, the leaf water potential of diseased vines increased progressively (i.e. showed less negative values), indicating a dysfunction in water and nutrient supply to the new growth. Both fungi were isolated from the xylem sap and from the woody tissue of branches and the trunk of diseased vines. Conidia isolated from the sap showed a high germination rate (>90%). Bioactive concentrations of the two pentaketides were detected in xylem sap, leaves and berries at various stages of seasonal growth. Exopolysaccharides, including pullulan, were found in the xylem sap. Absorption of culture filtrates of Pch and Tmi, as well as weak solutions of purified preparations of scytalone, isosclerone or pullulan, on detached leaves and berries caused symptoms similar to those shown by the esca-affected vines in the field.     

Effects of three esca-associated fungi on Vitis vinifera L.: V. Changes in the chemical and biological profile of xylem sap from diseased cv. Sangiovese vines

A vineyard of Vitis vinifera cv. Sangiovese was surveyed for incidence of esca and xylem sap collection. Sap samples were collected from healthy vines and from those with dual infection by Phaeomoniella chlamydospora (Pch) and Togninia minima (Tmi) or triple infection by Pch, Tmi and Fomitiporia mediterranea (Fme), during each early spring in a 3-year period (2001–2003). In order to analyse the possible trends in the climatic data, temperature and rainfall were assessed. At sap harvesting, aliquots of sap were assayed for phytotoxicity and extracted with ethyl acetate for phytotoxin recovery. Moreover, the exopolysaccharide (EPS) content was evaluated on several sap samples during the bleeding period. Conidia of Pch and Tmi, mycelium of Fme and their secondary metabolites were found in the sap of the esca-affected vines, indicating that the pathogens and their by-products together with some defence substances were accumulated and then translocated. Bioactivity tests showed toxicity of the sap from esca-affected vines to healthy detached leaves of cv. Sangiovese. The daily amount of sap, the pH, and the volume (J_v) and solute (J_s) fluxes were analysed as a function of the infecting fungi. Pullulan, glucogalactomannan(s) and arabinogalactan(s) are the main EPS in the esca-infected vines, whereas in the sap of healthy vines no traces of pullulan were found. Scytalone and isosclerone usually produced in vitro by Pch and Tmi were also detected in the sap of vines infected by Pch and Tmi or by Pch, Tmi and Fme. The endogenous phytohormone content of healthy vines evaluated by the cutting bioassay was different from that of infected vines. Four phenolics belonging to three classes e.g., benzoic acid derivatives, stilbenes and flavonol-glycosides were separated and identified by HPLC.     

Effects of three esca-associated fungi on Vitis vinifera L.: V. Changes in the chemical and biological profile of xylem sap from diseased cv. Sangiovese vines

A vineyard of Vitis vinifera cv. Sangiovese was surveyed for incidence of esca and xylem sap collection. Sap samples were collected from healthy vines and from those with dual infection by Phaeomoniella chlamydospora (Pch) and Togninia minima (Tmi) or triple infection by Pch, Tmi and Fomitiporia mediterranea (Fme), during each early spring in a 3-year period (2001–2003). In order to analyse the possible trends in the climatic data, temperature and rainfall were assessed. At sap harvesting, aliquots of sap were assayed for phytotoxicity and extracted with ethyl acetate for phytotoxin recovery. Moreover, the exopolysaccharide (EPS) content was evaluated on several sap samples during the bleeding period. Conidia of Pch and Tmi, mycelium of Fme and their secondary metabolites were found in the sap of the esca-affected vines, indicating that the pathogens and their by-products together with some defence substances were accumulated and then translocated. Bioactivity tests showed toxicity of the sap from esca-affected vines to healthy detached leaves of cv. Sangiovese. The daily amount of sap, the pH, and the volume (J_v) and solute (J_s) fluxes were analysed as a function of the infecting fungi. Pullulan, glucogalactomannan(s) and arabinogalactan(s) are the main EPS in the esca-infected vines, whereas in the sap of healthy vines no traces of pullulan were found. Scytalone and isosclerone usually produced in vitro by Pch and Tmi were also detected in the sap of vines infected by Pch and Tmi or by Pch, Tmi and Fme. The endogenous phytohormone content of healthy vines evaluated by the cutting bioassay was different from that of infected vines. Four phenolics belonging to three classes e.g., benzoic acid derivatives, stilbenes and flavonol-glycosides were separated and identified by HPLC.     

Effects of three esca-associated fungi on Vitis vinifera L.: III. Enzymes produced by the pathogens and their role in fungus-to-plant or in fungus-to-fungus interactions

When the esca-associated fungi Phaeomoniella chlamydospora (Pch), Togninia minima (Tmi) and Fomitiporia mediterranea (Fme) were grown in liquid stationary cultures, it was seen that they were able to live in media containing resveratrol (RES) or tannic acid (TA) as the sole carbon source and that the fungi were able to convert both compounds. Particular attention is paid here to detecting RES and TA conversion. Pch, Tmi and Fme were partially inhibited by RES or TA. Pch, Tmi and Fme produced extracellular tannase, laccase and peroxidase enzymes in liquid or agarized cultures, whether glucose was present or not. When colonies of Pch, Tmi and Fme were confronted, they showed spatially and temporally heterogeneous patterns of laccase and peroxidase activity. The results indicate the non-synergistic, competitive association of Pch and Tmi and the inhibition of Fme growth. Muconic acid, a well-known intermediate in a large number of lignin and phenol oxidative processes, can partly or completely inhibit the lignolytic agent Fme, but is tolerated by Pch and Tmi. An explanation for wood pigmentation patterns by Pch, Tmi and Fme is given.     

Esca in young and mature vineyards,and molecular diagnosis of the associated fungi

Esca is one of the most important grapevine trunk diseases, and it can induce severe decline. In the past, the disease occurred mostly on mature vines, but today it is also present in young vineyards. The aim of this study was to investigate the incidence of esca in young (< 7 years old) and mature (> 11 years old) vineyards on cvs Montepulciano, Sangiovese, Verdicchio and Passerina located in the main viticultural areas of the Marche Region, central-eastern Italy. The average incidence of diseased plants was higher in mature (32.6%) than young (5.2%) vineyards, and Verdicchio and Passerina appeared to be the most sensitive among the cultivars considered, followed by Sangiovese and Montepulciano. The analysis of the spatial spread of esca carried out in two mature vineyards on cv. Verdicchio and a young vineyard on cv. Sangiovese showed a fluctuation in the numbers of infected plants over the three years of observation. The fungi associated with symptomatic plants were detected by classical and molecular tools. Isolation on agar media yielded colonies of Phaeomoniella chlamydospora (Pch), Botryosphaeria spp. (Bot), Fomitiporia mediterranea (Fomed) and, sporadically, Phaeoacremonium aleophilum (Pal). In samples from young plants, Bot and Pch were recurrent, while Pch and Fomed were found in mature vines and old rootstocks. Molecular detection with specific primer pairs for Pch, Pal, Fomed, and B. dothidea confirmed the data obtained using classical tools, and in some cases it was more sensitive. This study thus provides a further contribution to the association between causal agents and esca symptoms, and it confirms the importance of molecular tools for a sensitive detection of associated pathogens, which can also be present in propagative materials.     

Analysis of head and leaf reaction towards <Emphasis Type="Italic">Microdochium nivale</Emphasis>

This research examined the variation in the response of eight commercial wheat cultivars to Microdochium nivale isolates using both in vivo FHB tests (AUDPC and RHW measurements) and in vitro detached leaf assays (LGR). Irrespective of fungal variety, the two Italian cvs Fortore and Norba exhibited the greatest amount of visual disease symptoms (mean AUDPC=2.2 and 2.3, respectively), being significantly more susceptible than the other six cultivars (AUDPC 1.24) (P < 0.05). Irrespective of fungal variety, the Italian cv. Norba and the Irish cv. Falstaff were more susceptible than the other cultivars (except Fatima 2) in terms of RHW (P < 0.05), while the cvs Fortore, GK Othalom and Consort were more resistant than the other five cultivars (P < 0.05). In the detached leaf assay, the Hungarian cv. GK Othalom and the Italian cv. Norba were more susceptible (mean LGR=0.79 and 0.81 mm day^–1, respectively) to M. nivalethan the other six cultivars (mean LGR=0.51–0.72) (P < 0.05). Analysis of the relationship between head and leaf reaction to M. nivaleinfection revealed no significant correlation.     

Host genotype- and temperature-dependent colonizationof In vitro carnation callus cultures byFusarium oxysporum f.sp.dianthi race 2

Differentin vivo resistance/susceptibility levels of 14 carnation cultivars toFusarium oxysporum f.sp.dianthi race 2, the causal agent of Fusarium wilt disease of carnation, were also expressed in anin vitro system and assayed as the degree of fungal colonization of callus cultures at 20° C. Temperature influenced thein vitro expression of carnation resistance. An incubation temperature of 27° C increased the colonization of calli derived from both the susceptible (‘Corrida’ and ‘Ambra’) and the resistant (‘Pulcino’ and ‘Pallas’) cultivars. At 15°C, the colonization of calli derived from Pulcino and Pallas diminished significantly more than for Ambra and Corrida. Inhibition of fungal growth on resistant calli was correlated to retardation in hyphal development. Both scanning electron microscopy and light microscopy observations showed that hyphae did not penetrate into carnation cells.     

Impact of alfalfa mosaic virus subgroup I and II isolates on terpene secondary metabolism of Lavandula vera D.C., Lavandula×alardii and eight cultivars of L. hybrida Rev.

Lavandula vera D.C., Lavandula×alardii and eight different L. hybrida Rev. cultivars cultivated at the Herb Garden of Casola Valsenio (Italy), found naturally infected by Alfalfa mosaic virus (AMV), have been evaluated for the influence of their pathologic status on essential oil biosynthesis and quality. The viral disease, consisting of a “yellow mosaic” on leaves and stems, was identified by means of electron microscopy, RT-PCR-RFLP and PAS-ELISA assays. Molecular characterization of the coat protein gene of four AMV isolates suggested that three of them belonged to subgroup I (from cvs. Ordinario and Grosso, Lavandula×alardii), while the isolate from cv. Sumiens showed molecular features of subgroup II. The infection of Lavandula plants by AMV (of the two subgroups) was found to decrease essential oil secretion. Changes in the relative composition regarded the linalool-linalyl acetate ratio; the overall increase in terpenic esters and in some cases increased sesquiterpenic abundance. Infection caused by AMV subgroup II isolate influenced negatively the quality of the essential oil much more than AMV isolates belonging to subgroup I. The importance of the phytopathological status of essential oil bearing crops is outlined.     

Control of Stagonospora nodorum and Septoria tritici in Wheat by Pre-treatment with Drechslera teres, a Non-host Pathogen

A field study is described which explored the possibility of controlling Stagonospora nodorum and Septoria tritici on wheat using a barley pathogen, Drechslera teres. Pre-treatment of wheat cv. Hussar flag leaves with D. teres resulted in a significant reduction in disease caused by S. nodorum and S. tritici, resulting in a significant increase in grain yield. When cv. Brigadier leaves were treated with D. teres prior to inoculation with S. nodorum there was an initial increase in disease expression whilst D. teres had no effect on symptoms produced by S. tritici on cv. Brigadier. There was significantly less disease on leaves of cvs. Hussar and Brigadier pre-treated with D. teres prior to inoculation with an equal mixture of S. nodorum and S. tritici compared to plants pre-treated with water. It is concluded that D. teres and other non-host pathogens show potential as biological control agents for S. nodorum and S. tritici.     

Symptom development, pathogen isolation and Real-Time QPCR quantification as factors for evaluating the resistance of olive cultivars to Verticillium pathotypes

Verticillium wilt is the most serious olive disease in the Mediterranean countries and worldwide. The most effective control strategy is the use of resistant cultivars. However, limited information is available about the level and source of resistance in most of the olive cultivars and there are no published data using microsclerotia, the resting structures of Verticillium dahliae, as the infective inoculum. In the present study, we correlated symptomatology and the presence of the fungus along with the DNA relative amount (molecules μl⁻¹) of a defoliating (D) and a non-defoliating (ND) V. dahliae strain in the susceptible cv. Amfissis and the tolerant cvs Kalamon and Koroneiki, as quantified by the Real-Time QPCR technology. The viability of the pathogen in the plant tissues was confirmed by isolating the fungus on PDA plates, while symptom assessment proved the correlation between the DNA relative amount of V. dahliae in plant tissues and cultivar susceptibility. It was further demonstrated that the D and ND strains were present at a significantly higher level in cv. Amfissis than in cvs Kalamon and Koroneiki. It was finally observed that the relative amount of the pathogen in roots was lower than in stems and shoots and declined in plant tissues over time. These data constitute a valuable contribution in evaluating resistance of olive cultivars or olive root-stocks to V. dahliae pathotypes.     

Production of phytotoxic metabolites by five species of Botryosphaeriaceae causing decline on grapevines, with special interest in the species Neofusicoccum luteum and N. parvum

In recent years an increasing number of species of Botryosphaeriaceae have been associated with grapevine decline worldwide. Five species isolated from declining grapevines in Spain (Botryosphaeria dothidea, Diplodia seriata, Dothiorella viticola, Neofusicoccum luteum and N. parvum) were checked for toxin production in liquid cultures. Cultural conditions for all fungi were adjusted to obtain optimal production of phytotoxic culture filtrates, by growing the fungi in steady liquid cultures of Czapek–Dox broth for different time intervals. Phytotoxicity of D. seriata and N. parvum reached a maximum after 14 days while the remaining species showed the highest phytotoxicity levels after 21 days in culture. All fungi produced hydrophilic high-molecular weight compounds with phytotoxic properties. In addition, N. luteum and N. parvum produced lipophilic low-molecular weight phytotoxins, not detected consistently among the remaining species. This led to a more exhaustive study on the phytotoxicity of N. luteum and N. parvum. Culture filtrates and corresponding extracts of both species were consistently highly phytotoxic in different assays. The gas-chromatography analysis of the acetylated O-methyl glycosides of the phytotoxic exopolysaccharides produced by N. parvum showed these substances to be composed mainly of glucose, mannose and galactose. Results suggest that phytotoxic metabolites could be involved in the virulence of both species in planta.     

Verbena as a trap crop to suppress thrips-transmitted Tomato spotted wilt virus in chrysanthemums

The effect of verbena as a trap crop on the occurrence of western flower thrips, Frankliniella occidentalis, and the incidence of Tomato spotted wilt virus (TSWV) in chrysanthemums were investigated. Verbena cvs. Pink Parfait and/or Fancy Parfait were cultivated alongside chrysanthemum cv. Jimba in a greenhouse in the proportion of 17%–25% of the chrysanthemum plants. Verbena plants attracted vector thrips, reducing western flower thrips colonization of chrysanthemum until flower bud initiation, and markedly suppressing TSWV incidence on chrysanthemums until flowering. Significant quantities of linalool oxide pyran were produced by the flower of cv. Fancy Parfait; and the ratio of cis-linalool oxide pyran, an attractant for vector thrips, to the trans-type was approximately 1 : 5. Our results suggest that cultivation of verbena as a trap crop may be useful in integrated pest management programs as a control for thrips-transmitted TSWV in chrysanthemums.     

Occurrence, isolation and biological activity of phytotoxic metabolites produced in vitro by Sphaeropsis sapinea, pathogenic fungus of Pinus radiata

Sphaeropsis sapinea was repeatedly isolated in Sardinia from symptomatic samples of the upper part of declining pine (Pinus radiata) plants. Observed symptoms mainly consisted of foliage chlorosis, drying of needles and cankers on branches. The S. sapinea strains were shown to produce phytotoxic metabolites in culture filtrates. Three metabolites were isolated for the first time from this fungus and identified by their spectroscopic and optical properties as R-(−)-mellein, (3R,4R)-4-hydroxymellein and (3R,4S)-4-hydroxymellein. When assayed for phytotoxic and antifungal activities on host and non-host plants and on some phytopathogenic fungi, the R-(−)-mellein showed significant activity, while the other two 3,4-dihydroisocoumarins showed only a synergic activity in both tests.     

Scab Response and Moniliformin Accumulation in Kernels of Oat Genotypes Inoculated with Fusarium avenaceum in Poland

Inoculation experiments with 14 genotypes of oats (10 cultivars and 4 lines) were performed during 1996, 1997 and 1998 in Sitaniec, South-Eastern Poland. Panicles of oats were inoculated with a conidial suspension of Fusarium avenaceum, which caused a reduction in yield by 33% and in 1000 kernel weight (TKW) by 21%. During the period between inoculation and harvest, F. avenaceum was able to accumulate moniliformin (MON) in kernels at an average level of 0.13mgkg^–1 (gg^–1). The highest reduction of yield components caused by the F. avenaceum inoculation was found for cv. Santor, followed by lines CHD 1171, STH 2795 and cvs: Kwant and Farys, while cvs Slawko, Dukat, Borys and Komes exhibited the highest resistance to the disease in terms of TKW and yield reductions after inoculation.     

一株生防内生真菌的分离筛选、鉴定及抑菌特性

为获取优良植物病害生防菌,以交链孢菌Alternaria tenuis Ness和尖镰孢菌Fusarium oxysporum Schlecht为指示菌,采用平板对峙培养法、抑菌圈法和菌落直径法对健康龙柏鳞叶小枝中分离的4株优势内生真菌进行了筛选评价,并结合形态学观察及ITS序列分析对筛选出的生防内生真菌进行了鉴定。结果表明,对峙培养发现1菌株对交链孢菌和尖镰孢菌均具有较好的抑制作用,抑菌率分别为52.05%和48.65%,标记其为LB-1;LB-1菌株与指示菌对峙培养的菌落交界面处,2种指示菌的菌丝均出现了消解、断裂,而LB-1的菌丝则生长正常。未灭菌LB-1菌株发酵液对交链孢菌和尖镰孢菌产生的抑菌圈直径分别为14.1 mm和13.7 mm,抑菌率分别为47.89%和48.72%;灭菌LB-1菌株发酵液对交链孢菌和尖镰孢菌的抑菌圈直径分别为15.3 mm和15.0 mm,抑菌率分别为5.96%和33.90%,指示菌在生长过程中菌丝溶解现象明显。经鉴定,LB-1菌株为毛壳菌属Chaetomium中的近缘毛壳菌C.subaffine,是一种极具生防潜力的内生真菌,其抑菌特性可能通过溶菌作用实现。