<Emphasis Type="Italic">Trichoderma harzianum</Emphasis> elicits defence response genes in roots of potato plantlets challenged by <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Biological control of Rhizoctonia solani with Trichoderma harzianum has been demonstrated in several studies. However, none have reported the dynamics of expression of defence response genes. Here we investigated the expression of these genes in potato roots challenged by R. solani in the presence/absence of T. harzianum Rifai MUCL 29707. Analysis of gene expression revealed an induction of PR1 at 168 h post-inoculation (hpi) and PAL at 96 hpi in the plants inoculated with T. harzianum Rifai MUCL 29707, an induction of PR1, PR2 and PAL at 48 hpi in the plants inoculated with R. solani and an induction of Lox at 24 hpi and PR1, PR2, PAL and GST1 at 72 hpi in the plants inoculated with both organisms. These results suggest that in the presence of T. harzianum Rifai MUCL 29707, the expression of Lox and GST1 genes are primed in potato plantlets infected with R. solani at an early stage of infection. Mycothèque de l’Université catholique de Louvain of S. Cranenbrouck's affiliation is part of the Belgian Coordinated Collections of Micro-organisms (BCCM).     

Increased susceptibility of potato to <Emphasis Type="Italic">Rhizoctonia</Emphasis> diseases in <Emphasis Type="Italic">Potato leafroll virus</Emphasis>-infected plants

In Hokkaido potato fields, tubers produced from the plants with leaf curl symptoms caused by potato leaf roll virus (PLRV) were noted to be more densely covered with Rhizoctonia sclerotia. This observation led us to hypothesize that potato infected with PLRV would have an increased susceptibility to Rhizoctonia solani. To test this hypothesis, in a pot experiment, we inoculated PLRV-infected mother tubers with Rhizoctonia. As a result, PLRV-infected plants produced significantly fewer and smaller tubers than virus-free plants did, suggesting that PLRV-infected plants are more susceptible than virus-free plants to R. solani. Virus-free seed tubers should thus be used to reduce Rhizoctonia diseases.     

BABA effects on the behaviour of potato cultivars infected by <Emphasis Type="Italic">Phytophthora infestans</Emphasis> and <Emphasis Type="Italic">Fusarium solani</Emphasis>

Since most plants possess resistance mechanisms which can be induced upon pre-treatment with a variety of chemical compounds, the use of β-aminobutyric acid (BABA) as a defence inducer without reported toxic effect on the environment was studied. The aim of this work was to analyse the effectiveness of BABA to induce resistance against Phytophthora infestans and Fusarium solani in potato cultivars differing in their level of resistance to late blight. The behaviour of some components of biochemical mechanisms by which BABA increases resistance against P. infestans, as well as the effect of BABA on the activity of a potential pathogenic factor of F. solani, were studied. Plants with four applications of BABA throughout the crop cycle produced tubers more resistant to P. infestans and F. solani than non-treated plants. In addition, tuber slices from treated plants, inoculated with P. infestans, showed an increase in phenol and phytoalexin content. The aspartyl protease StAP1 accumulation was also higher in tubers obtained from treated plants and inoculated with P. infestans. This result was observed only in the more resistant potato cv. Pampeana, early after infection. In the potato–F. solani interaction, infected tubers coming from BABA-treated plants showed minor fungal proteolytic activity than infected, non-treated ones. For potato cvs Pampeana and Bintje, the BABA treatment improved the yield of harvested tubers. The number of tubers per plant and total weight of harvested tubers was greater for those obtained from treated plants with two early or four applications of BABA. The results show that the BABA treatment increases the resistance of potatoes but the degree of increase depends on the original level of resistance present in each cultivar.     

Identification of <Emphasis Type="Italic">Globodera rostochiensis</Emphasis> and <Emphasis Type="Italic">G. pallida</Emphasis> in the Ukraine by PCR

Multiplex polymerase chain reaction was used to identify the potato cyst nematodes in soil samples from the Ukraine. The results show the occurrence of Globodera pallida in the Uzhhorod region (Zakarpatska oblast), where only G. rostochiensis had been previously reported. In the mixed potato cyst nematode (PCN) populations, G. pallida was less prevalent (2–5%) than G. rostochiensis (95–98%). A phylogenetic analysis based on ribosomal DNA internal transcribed spacer sequences showed that the Ukrainian population of G. pallida had >99% sequence identity with other G. pallida pa2/3 isolates from Europe. This study has demonstrated that polymerase chain reaction-mediated amplification of specific regions of the potato cyst nematode genome is not only highly effective as a species diagnostic tool but is also a sensitive method which can be used for taxonomic purposes with cyst collections which vary in age.     

Inheritance of resistance in <Emphasis Type="Italic">Solanum nigrum</Emphasis> to <Emphasis Type="Italic">Phytophthora infestans</Emphasis>

Solanum nigrum, black nightshade, is a wild non-tuber bearing hexaploid species with a high level of resistance to Phytophthora infestans (Colon et al. 1993), the causal agent of potato late blight, the most devastating disease in potato production. However, the genetic mode of resistance in S. nigrum is still poorly understood. In the present study, two S. nigrum accessions, 984750019 (N19) and #13, resistant (R) and susceptible (S), respectively, to three different isolates of P. infestans, were sexually crossed. The various kinds of progeny including F1, F2, F3, and backcross populations (BC₁; F1 × S), as well as two populations produced by self-pollinating the R parent and S parent, were each screened for susceptibility to P. infestans isolate MP 324 using detached leaf assays. Fifty seedling plant individuals of the F1 progeny were each resistant to this specific isolate, similarly to the seedling plants resulting from self-pollination of the resistant R parent. Thirty seedling plants obtained from self-pollination of the S parent were susceptible. Among a total of 180 F2 plants, the segregation ratio between resistant and susceptible plants was approximately 3: 1. Among the 66 seedling plants of the BC₁ progeny originating from crossing an F1 plant with the susceptible S parent, there were 26 susceptible and 40 resistant plants to P. infestans. The segregation patterns obtained indicated monogenic dominant inheritance of resistance to P. infestans isolate MP 324 in S. nigrum acc. 984750019. This gene, conferring resistance to P. infestans, may be useful for the transformation of potato cultivars susceptible to late blight.     

Cytomolecular aspects of rice sheath blight caused by <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Sheath blight, caused by anastomosis group 1-IA of Rhizoctonia solani Kühn (teleomorph Thanatephorus cucumeris (Frank) Donk), is one of the most destructive rice diseases worldwide. The pathogen is able to infect plants belonging to more than 27 families, including many economically important monocots and dicots such as rice, wheat, alfalfa, bean, peanut, soybean, cucumber, papaya, corn, potato, tomato and sugar beet. It is a soil borne necrotrophic fungus that survives in plant debris as sclerotia, which are small brown-to-black, rocklike reproductive structures. The sclerotia can survive in the soil for several years and infect rice plants at the water-plant interface in the flooded field by producing mycelia. Management of rice sheath blight requires an integrated approach based on the knowledge of each stage of the disease and cytomolecular aspects of rice defence responses against R. solani. This review summarizes current knowledge on molecular aspects of R. solani pathogenicity, genetic structure of the pathogen populations, and the rice-R. solani interaction with emphasis on cellular and molecular defence components such as signal transduction pathways, various plant hormones, host defence genes and production of defence-related proteins involved in basal and induced resistance in rice against sheath blight disease.     

The use of GFP<Emphasis Type="Italic">-</Emphasis>transformed isolates to study infection of banana with <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">cubense</Emphasis> race 4

Fusarium oxysporum f. sp. cubense (Foc) is the causal pathogen of Fusarium wilt of banana. To understand infection of banana roots by Foc race 4, we developed a green fluorescent protein (GFP)-tagged transformant and studied pathogenesis using fluorescence microscopy and confocal laser scanning microscopy. The transformation was efficient, and GFP expression was stable for at least six subcultures with fluorescence clearly visible in both hyphae and spores. The transformed Foc isolate also retained its pathogenicity and growth pattern, which was similar to that of the wild type. The study showed that: (i) Foc race 4 was capable of invading the epidermal cells of banana roots directly; (ii) potential invasion sites include epidermal cells of root caps and elongation zone, and natural wounds in the lateral root base; (iii) in banana roots, fungal hyphae were able to penetrate cell walls directly to grow inside and outside cells; and (iv) fungal spores were produced in the root system and rhizome. To better understand the interaction between Foc race 4 and bananas, nine banana cultivars were inoculated with the GFP-transformed pathogen. Root exudates from these cultivars were collected and their effect on conidia of the GFP-tagged Foc race 4 was determined. Our results showed that roots of the Foc race 4-susceptible banana plants were well colonized with the pathogen, but not those of the Foc race 4-resistant cultivars. Root exudates from highly resistant cultivars inhibited the germination and growth of the Fusarium wilt pathogen; those of moderately resistant cultivars reduced spore germination and hyphal growth, whereas the susceptible cultivars did not affect fungal germination and growth. The results of this work demonstrated that GFP-tagged Foc race 4 isolates are an effective tool to study plant–fungus interactions that could potentially be used for evaluating resistance in banana to Foc race 4 by means of root colonization studies. Banana root exudates could potentially also be used to identify cultivars in the Chinese Banana Germplasm Collection with resistance to the Fusarium wilt pathogen.     

<Emphasis Type="Italic">Trichoderma harzianum</Emphasis>- mediated reprogramming of oxidative stress response in root apoplast of sunflower enhances defence against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Trichoderma harzianum is an effective biocontrol agent against the devastating plant pathogen Rhizoctonia solani. Despite its wide application in agriculture, the mechanisms of biocontrol are not yet fully understood. Mycoparasitism and antibiosis are suggested, but may not be sole cause of disease reduction. In the present study, we investigated the role of oxidant-antioxidant metabolites in the root apoplast of sunflower challenged by R. solani in the presence/absence of T. harzianum NBRI-1055. Analysis of oxidative stress response revealed a reduction in hydroxyl radical concentration (^•OH; 3.6 times) at 9 days after pathogen inoculation (dapi), superoxide anion radical concentration (O₂^•−; 4.1 times) at 8 dapi and hydrogen peroxide concentration (H₂O₂; 2.7 times) levels at 7 dapi in plants treated with spent maize-cob formulation of T. harzianum NBRI-1055 (MCFT), as compared to pathogen-inoculated plants. The protection afforded by the biocontrol agent was associated with the accumulation of the ROS gene network: the catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and ascorbate peroxidase (APx), maximum activity of CAT (11.0 times) was observed at 8 dapi, SOD (7.0 times) at 7 dapi, GPx (5.4 times) and APx (8.1 times) at 7 dapi in MCFT-treated plants challenged with the pathogen. This was further supported by the inhibition of lipid and protein oxidation in Trichoderma-inoculated plants. MCFT stimulated the accumulation of secondary metabolites of phenolic nature that increased up to five-fold and also exhibited strong antioxidant activity at 8 dapi, eventually leading to the systemic accumulation of phytoalexins. These results suggest that T. harzianum–mediated biocontrol may be related to alleviating R. solani-induced oxidative stress.     

A fungistatic chromene from<Emphasis Type="Italic">Ageratum conyzoides</Emphasis>

Ageratum conyzoides L. is an annual herb in the tropics and subtropics whose extracts are known to possess pharmacological and biocidal activity. We report on the bioactivity of a secondary metabolite (a chromene) isolated from the shoots ofA. conyzoides against some plant pathogenic fungi. Organic solvent extracts from the shoots were tested for antifungal activity against the plant pathogenic fungiRhizoctonia solani, Sclerotium rolfsii, Botryodiplodia theobromae, Phomopsis theae andFusarium species growingin vitro on potato dextrose agar medium. The cruden-hexane extract completely inhibited the growth ofR. solani andS. rolfsii. Then-hexane extract was chromatographed over a column of silica gel followed by activity-guided fractionation to give an antifungal principle. Structure elucidation by detailed analysis of¹H,¹³C NMR and mass spectroscopy identified the active compound as precocene II. The growth ofR. solani andS. rolfsii was completely inhibited by precocene II at a concentration of 80–100 ppm. The sclerotia ofR. solani andS. rolfsii were also completely suppressed by 150 ppm of precocene II. Sub-culture of these inhibited fungi onto precocene II-free medium restored growth of the fungus, indicating that precocene II is fungistatic. Crude or refined extracts fromA. conyzoides offer the possibility of biocontrol of plant pathogenic fungi. http://www.phytoparasitica.org posting Feb. 11, 2004.     

Potato <Emphasis Type="Italic">R1</Emphasis> resistance gene confers resistance against <Emphasis Type="Italic">Phytophthora infestans</Emphasis> in transgenic tomato plants

Tomato is challenged by several pathogens which cause loss of production. One such pathogen is the oomycete Phytophthora infestans which is able to attack all the aerial parts of the plant. Although a wide range of resistance sources are available, genetic control of this disease is not yet successful. Pyramiding R-genes through genetic transformation could be a straightforward way to produce tomato and potato lines carrying durable resistance to P. infestans. In this work the R1 potato gene was transferred into tomato lines. The tomato transgenic lines were analyzed by using q-RT-PCR and progeny segregation to determine the gene copy number. To test the hypothesis that R1 represents a specifically regulated R-gene, transgenic tomato plants were inoculated with P. infestans isolate 88133 and IPO. All the plants containing the R1 gene were resistant to the late blight isolate IPO-0 and susceptible to isolate 88133. These results provide evidence for specific activation of the R1 gene during pathogen challenge. Furthermore, evidence for enhancement of PR-1 gene expression during P. infestans resistance response was obtained.     

Pathogenicity and reproductive fitness of <Emphasis Type="Italic">Pratylenchus coffeae</Emphasis> and <Emphasis Type="Italic">Radopholus arabocoffeae</Emphasis> on Arabica coffee seedlings (<Emphasis Type="Italic">Coffea arabica</Emphasis> cv. Catimor) in Vietnam

The pathogenicity and reproductive fitness of Pratylenchus coffeae and Radopholus arabocoffeae from Vietnam on coffee (Coffea arabica) seedlings cv. Catimor were evaluated in greenhouse experiments. The effect of initial population densities (Pi = 0, 1, 2, 4, 8, 16, 32, 64, 128, and 256 nematodes per cm³ soil) was studied for both species at different days after inoculation (dai). The data were adjusted to the Seinhorst damage model Y = m + (1-m).z^Pi-T. Tolerance limit (T) for P. coffeae was zero for the height and the diameter of the coffee plants. For the diameter, the T-value for R. arabocoffeae was 25.6 for 30 and 60 dai and 12.8 for 90 and 120 dai. After 4 months T was zero. The low tolerance limits indicate that Arabica coffee is highly intolerant to both nematode species. At the end of the experiment (180 dai), all plants were infected and most were dead when inoculated with R. arabocoffeae at initial densities of 32, 64, 128 and 256 nematodes/cm³ soil. For P. coffeae plant death was already observed at the lowest inoculation densities. Growth of coffee was reduced at all inoculation levels for both species. Pratylenchus coffeae and R. arabocoffeae caused intense darkening of the roots, leaf chlorosis and a strong reduction of root and shoot growth. It was observed that P. coffeae mainly destroyed lateral roots rather than tap roots, whereas R. arabocoffeae reduced tap root length rather than the lateral roots. At the lowest inoculum densities, the reproduction factor of P. coffeae was 2.38 and 2.01 for R. arabocoffeae, indicating that arabica coffee is a host for both species. Plant growth as expressed by shoot height and shoot and root weight measured 60 dai was negatively correlated with nematode (both species) density as expressed by the geometric mean of nematode numbers at 30 and 60 dai.     

Germination of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis> resting spores stimulated by a non-host plant

Plant-induced germination of Plasmodiophora brassicae resting spores was studied in a laboratory experiment. Spore reaction was analysed in nutrient solution with exudates from growing roots of different plant species – one host plant (Brassica rapa var. pekinensis) and four non-host plants (Lolium perenne, Allium porrum, Secale cereale and Trifolium pratense) – and in controls with distilled water and nutrient solution. It was found that root exudates from L. perenne stimulated spore germination more than exudates from the other plants, including those from the host plant. The effect could not be explained by differences in the nutritional composition of the solutions due to differential uptake of the plant species, or by differences in root activity, measured as exudation of soluble sugars. This is the first time such a separation of factors has been done in analysing the influence of plants on P. brassicae germination. Although stimulation of P. brassicae resting spore germination is not restricted to the presence of host plants, it seems to vary depending on the plant species.     

Antifungal activities of hyoscyamine and scopolamine against two major rice pathogens: <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> and <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The antifungal activities of hyoscyamine and scopolamine, major alkaloids extracted from the desert plant Hyoscyamus muticus, against two rice pathogens, Magnaporthe oryzae and Rhizoctonia solani, were studied. The minimum inhibitory concentration of hyoscyamine that resulted in distinctive inhibition (MIC₅₀) was 1 μg/ml for both fungi. Exposure to hyoscyamine caused the leakage of electrolytes from the mycelia of both fungi. Hyoscyamine (>1 μg/ml) irreversibly delayed or inhibited conidial germination and appressorium formation in M. oryzae grown on polystyrene plates. Hyoscyamine effectively inhibited the attachment of conidia to the surface of rice (Oryza sativa) leaves and inhibited appressorium formation on the leaves. A high concentration of scopolamine (1000 μg/ml) also delayed or inhibited conidial germination in M. oryzae, but conidial germination was restored after washing the conidia with water. Antifungal activity of hyoscyamine was reduced by scopolamine. Magnaporthe oryzae infection was significantly suppressed (by >95%) in leaves of intact rice plants treated with hyoscyamine (10 μg/ml). Moreover, 10 μg hyoscyamine/ml significantly reduced the disease severity index for sheath blight to ≤0.2, when compared with the disease index of control plants (>7.0). Hyoscyamine (>20 μg/ml) completely inhibited sclerotial germination and development of R. solani by delaying the initiation, maturation, and melanization of the sclerotia. These results suggest that tropane alkaloids may be useful for controlling blast and sheath blight diseases of rice and for studying the mechanisms that regulate conidial germination in M. oryzae and sclerotial germination and development in R. solani.     

Bacterial wilt of sweet potato caused by <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in Taiwan

During the last decade, a new bacterial disease has impaired the yield of vegetable sweet potato (30–80%) in Taiwan. Infected plants developed stunting, root and stem rot, vascular discoloration and wilting. Ten bacterial isolates that caused the same symptoms in sweet potatoes after inoculation were reisolated and classified as Ralstonia solanacearum phylotype I biovar 4 based on physical and molecular analyses. Moreover, these isolates also caused wilting in convolvulaceous, solanaceaous and cruciferous plants. This report is the first of bacterial wilt of sweet potato caused by R. solanacearum in Taiwan.     

<Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> can cause potato blackleg in temperate climates

It is well established that the pectinolytic bacteria Pectobacterium atrosepticum (Pca) and Dickeya spp. are causal organisms of blackleg in potato. In temperate climates, the role of Pectobacterium carotovorum subsp. carotovorum (Pcc) in potato blackleg, however, is unclear. In different western and central European countries plants are frequently found with blackleg from which only Pcc can be isolated, but not Pca or Dickeya spp. Nevertheless, tubers vacuum-infiltrated with Pcc strains have so far never yielded blackleg-diseased plants in field experiments in temperate climates. In this study, it is shown that potato tubers, vacuum-infiltrated with a subgroup of Pcc strains isolated in Europe, and planted in two different soil types, can result in up to 50% blackleg diseased plants.     

Genetic diversity of <Emphasis Type="Italic">Rhizobium vitis</Emphasis> strains in Japan based on multilocus sequence analysis of <Emphasis Type="Italic">pyrG</Emphasis>, <Emphasis Type="Italic">recA</Emphasis> and <Emphasis Type="Italic">rpoD</Emphasis>

Forty-one strains of Rhizobium vitis, either tumorigenic (Ti) or nonpathogenic, were characterized using multilocus sequence analysis (MLSA) of the partial nucleotide sequences of pyrG, recA, and rpoD. The strains separated into seven clades. Rhizobium vitis (Ti) strains isolated from Japan were divided into five genetic groups (A to E), and nonpathogenic R. vitis strains were divided into two genetic groups (F and G). This result suggests that there are new genetic groups of R. vitis in Japan. Among these groups, members of A and B groups are widely distributed throughout Japan.     

Induced resistance against Fusarium diseases of <Emphasis Type="Italic">Cymbidium</Emphasis> species by weakly virulent strain HPF-1 (<Emphasis Type="Italic">Fusarium</Emphasis> sp.)

The mechanism by which Fusarium diseases of cymbidium plants are suppressed by a weakly virulent strain HPF-1 of Fusarium sp. was studied. Strain HPF-1 produced microscopic, necrotic local lesions on cymbidium leaves, causing minor damage to palisade tissues at the infection sites. This weakly virulent strain remained near the site of infection and did not develop further. It systemically and nonselectively suppressed some diseases of cymbidium such as yellow spot of leaves caused by Fusarium proliferatum and F. fractiflexum, bulb and root rot caused by F. oxysporum, and dry rot of bulbs and roots caused by F. solani. Because endogenous salicylic acid levels increased in cymbidium leaves inoculated with strain HPF-1, the mechanism of disease suppression is thought to be systemic acquired resistance.     

Nematicidal potential of materials from <Emphasis Type="Italic">Medicago</Emphasis> spp.

The nematicidal effect of soil amendments with dry top and root material from Medicago sativa and/or Medicago arborea was evaluated on the root-knot nematode Meloidogyne incognita and on the cyst nematode Globodera rostochiensis in potting mixes. All amendments suppressed root and soil population densities of both nematode species compared to non-treated and chemical controls. The suppressiveness of M. sativa differed between top and root material and among the amendment rates. In field conditions soil amendments with 20 or 40 t ha⁻¹ of a pelleted M. sativa meal increased tomato crop yield and reduced soil population densities and root galling by M. incognita. It is suggested that saponins were at least partly responsible for the nematicidal activity.     

Epidemiology of root rot caused by <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> in <Emphasis Type="Italic">Brassica napus</Emphasis> crops

The infection of above-ground tissues of Brassica napus by Leptosphaeria maculans is well understood. However, root infection (root rot) under field conditions, the development of root rot over time and its relationship to other disease symptoms caused by L. maculans has not been described. A survey of B. napus crops was conducted in Australia to investigate the incidence and severity of root rot. Additionally, the pathway of root infection was examined in field experiments. Root rot was present in 95% of the 127 crops surveyed. The severity and incidence of root rot was significantly correlated with that of crown canker; however, the strength of this relationship was dependent on the season. Root rot symptoms appeared before flowering and increased in severity during flowering and at maturity, a pattern similar to crown canker suggesting that the infection of the root is an extension of the crown canker phase of the L. maculans lifecycle. All isolates of L. maculans tested in glasshouse experiments caused root rot and crown canker in B. napus and Brassica juncea. In the field, the main pathway of root infection is via invasion of cotyledons or leaves by airborne ascospores, rather than from inoculum in the soil. Root rot was present in crops in fields that had never been sown to B. napus previously, in plants grown in fumigated fields, and in glasshouse-grown plants inoculated in the hypocotyl with L. maculans.