Primary structure of cytochrome c gene from the white root rot fungus Rosellinia necatrix

The nucleotide sequence of the cytochrome c (CytC) gene of the white root rot fungus Rosellinia necatrix was analyzed. The structure of this gene, which had three introns in the coding region, was similar to that of Aspergillus nidulans. The second intron of the R. necatrix CytC gene was not present in Neurospora crassa or Fusarium oxysporum. However, the amino acid sequence of R. necatrix was most similar to that of Neurospora crassa. Thus, it seemed that the second intron of the R. necatrix CytC gene was inserted into its present position after R. necatrix and its closest relatives diverged evolutionarily.     

Comparison between Rosellinia necatrix isolates from soil and diseased roots in terms of hypovirulence

The white root rot fungus, Rosellinia necatrix, is a devastating soil-borne pathogen of many plant species. Biocontrol with the hypovirulence factor is promising, but disease symptoms, signs or culture morphology of the pathogen cannot be reliably used as markers for hypovirulence in this fungus. We attempted to obtain hypovirulent isolates from soil rather than from diseased roots, based on the hypothesis that hypovirulent isolates were more likely to persist in soil as saprobes. Sixteen isolates, belonging to eight mycelial compatibility groups (MCGs), were obtained from soil in two active and one abandoned Japanese pear orchards. Comparison of these isolates based on clonality revealed that six MCGs were commonly recovered from both diseased roots and soil and two MCGs exclusively from soil. No MCG was found in more than one orchard. With two exceptions, isolates within the same MCG were similar in virulence, competitive saprophytic ability (CSA) and mycelial growth rate whether or not they carried dsRNA. The two exceptional isolates recovered from soil had multiple dsRNA segments that caused hypovirulence, weakened CSA and restricted mycelial growth on nutrient-rich media. They belonged to different MCGs, each including dsRNA-free isolates. Isolates from soil contained various dsRNAs (44%), including the hypovirulence factor, more frequently than isolates from diseased roots in the same fields (25%), which is much higher than the proportion of isolates with dsRNA from diseased roots (19%) in a total of 424 isolates from Japan examined so far. These results suggest that isolation of R. necatrix from soil is an effective method to obtain isolates with dsRNAs, including the hypovirulence factor.     

BROWN ROOT ROT OF TOMATOES

Tomato roots with brown root rot showed three types of lesion: cortical rot of fine roots, 'corky root', and basal stem rot. The fungi most commonly isocated from diseased roots were: Colletotrichum atramentarium, Chaetomium spp., Cephalosporium spp., Volutella ciliata , and a grey mycelial fungus sometimes producing pycnidia ( Pyrenochaeta sp.). Other fungi isocated less frequently were: Myrothecium roridum, Petriella asymmetrica, Trichoderma airide, Phytophthora spp., Alternaria sp. and Fusarium spp. There were differences between the numbers of each species isocated from the three types of lesion in steamed and in unsteamed soils, and some seasonal variation.
Inoculation experiments with seedlings in vitro showed that Ckaetomium cochliodes and Petriella asymmetrica could infect radicles. On older plants growing in soil Colletotrichum atramentarium was the only effective pathogen. Culture filtrates from C. atramentarium, Chaetomium cochliodes , and P. asymmetrica decreased root growth when tested in vitro. Leachates from loam and a loam-manure mixture decreased the growth of tomato root-tip cultures; the effect of these leachates was altered by autoclaving.     

Tobacco leaf spot and root rot caused by Rhizoctonia solani Kühn

Rhizoctonia solani Kühn is a soil-borne fungal pathogen that causes disease in a wide range of plants worldwide. Strains of the fungus are traditionally grouped into genetically isolated anastomosis groups (AGs) based on hyphal anastomosis reactions. This article summarizes aspects related to the infection process, colonization of the host and molecular mechanisms employed by tobacco plants in resistance against R. solani diseases. TAXONOMY: Teleomorph: Thanatephorus cucumeris (Frank) Donk; anamorph: Rhizoctonia solani Kühn; Kingdom Fungi; Phylum Basidiomycota; Class Agaricomycetes; Order Cantharellales; Family Ceratobasidiaceae; genus Thanatephorus. IDENTIFICATION: Somatic hyphae in culture and hyphae colonizing a substrate or host are first hyaline, then buff to dark brown in colour when aging. Hyphae tend to form at right angles at branching points that are usually constricted. Cells lack clamp connections, but possess a complex dolipore septum with continuous parenthesomes and are multinucleate. Hyphae are variable in size, ranging from 3 to 17 μm in diameter. Although the fungus does not produce any conidial structure, ellipsoid to globose, barrel-shaped cells, named monilioid cells, 10-20 μm wide, can be produced in chains and can give rise to sclerotia. Sclerotia are irregularly shaped, up to 8-10 mm in diameter and light to dark brown in colour. DISEASE SYMPTOMS: Symptoms in tobacco depend on AG as well as on the tissue being colonized. Rhizoctonia solani AG-2-2 and AG-3 infect tobacco seedlings and cause damping off and stem rot. Rhizoctonia solani AG-3 causes 'sore shin' and 'target spot' in mature tobacco plants. In general, water-soaked lesions start on leaves and extend up the stem. Stem lesions vary in colour from brown to black. During late stages, diseased leaves are easily separated from the plant because of severe wilting. In seed beds, disease areas are typically in the form of circular to irregular patches of poorly growing, yellowish and/or stunted seedlings. RESISTANCE: Knowledge is scarce regarding the mechanisms associated with resistance to R. solani in tobacco. However, recent evidence suggests a complex response that involves several constitutive factors, as well as induced barriers controlled by multiple defence pathways. MANAGEMENT: This fungus can survive for many years in soil as mycelium, and also by producing sclerotia, which makes the management of the disease using conventional means very difficult. Integrated pest management has been most successful; it includes timely fungicide applications, crop rotation and attention to soil moisture levels. Recent developments in biocontrol may provide other tools to control R. solani in tobacco.     

Study on Rosellinia necatrix Isolates Causing White Root Rot Disease in Taiwan

White root rot is a serious soil‐borne disease of several woods and crops. Recently, white root rot of tea shrubs and ornamental trees has increasingly been observed in Taiwan. Thirty‐six isolates of white root rot pathogen, showing pear‐shape swellings adjacent to the hyphal septa, had been isolated from samples of white root rot collected from Taiwan for about 4 years. The pathogen isolates produced Dematophora anamorph. Conidia of the pathogen were one‐celled, hyaline, subglobal, with truncate base, 2.9–5.8 × 1.9–3.5 μm . Ascospore dimensions were in the range of 37.0–55.0 × 5.4–7.9 μm with a short, longitudinal and straight germ slit, which complied with Rosellinia necatrix. Based on molecular studies, the pathogen isolates collected from Taiwan except R701 were identified as R. nectarix. Isolate R701, which was relatively polymorphic in internal transcribed spacer DNA sequence than other isolates, was temporarily considered as R. necatrix‐related pathogenic Rosellinia spp. All the tea cuttings (Camellia sinensis) inoculated with isolates developed typical white root rot symptoms. Pathogenicity tests demonstrated the presence of variation in virulence among the Rosellina isolates. Most of the R. necatrix isolates originating from Acer morrisonense were less virulent than those that originated from other hosts. The pathogenic Rosellinia spp., isolate R701, was also highly virulent to both cultivars of tea cuttings.     

Improved real-time PCR protocol for the accurate detection and quantification of Rosellinia necatrix in avocado orchards

Plant and Soil - This study aims to develop and validate a new molecular method of detection and quantification of Rosellinia necatrix fungus in soil samples and compare it with conventional...     

A Review of the Biology and Pathogenicity of Rosellinia necatrix– The Cause of White Root Rot Disease of Fruit Trees and Other Plants

Rosellinia necatrix, an ascomycete soil‐inhabiting fungus, causes white root rot disease in a large number of plant species, especially fruit trees. The fungus, which occurs worldwide, is very aggressive and can kill infected trees. The biology and pathogenicity of the fungus are reviewed here, together with the current principal methods of disease control used in different pathosystems.     

Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus Rosellinia necatrix

Rosellinia necatrix is a soil-borne root pathogen affecting a wide range of commercially important plant species. The mycelium of R. necatrix was transformed to hygromycin B resistance by an Agrobacterium tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) gene controlled by the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. Co-cultivation of R. necatrix strain W1015 and A. tumefaciens strain AGL-1 at 25 degrees C using the binary vector pAN26-CB1300, which contained the hygromycin B resistance cassette based on pAN26 and pCAMBIA1300, resulted in high frequencies of transformation. The presence of the hph gene in the transformants was detected by PCR, and single-copy integration of the marker gene was demonstrated by Southern blot analysis. This report of an Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system f or insertional mutagenesis in R necatrix and provide a simple and reliable method for genetic manipulation.     

Two similar enhanced root-colonizing Pseudomonas strains differ largely in their colonization strategies of avocado roots and Rosellinia necatrix hyphae

Pseudomonas alcaligenes AVO73 and Pseudomonas pseudoalcaligenes AVO110 were selected previously as efficient avocado root tip colonizers, displaying in vitro antagonism towards Rosellinia necatrix, causal agent of avocado white root rot. Despite the higher number of antagonistic properties shown in vitro by AVO73, only AVO110 demonstrated significant protection against avocado white root rot. As both strains are enhanced root colonizers, and as colonization is crucial for the most likely biocontrol mechanisms used by these strains, namely production of non-antibiotic antifungal compounds and competition for nutrients and niches, we decided to compare the interactions of the bacterial strains with avocado roots as well as with R. necatrix hyphae. The results indicate that strain AVO110 is superior in biocontrol trait swimming motility and establishes on the root tip of avocado plants faster than AVO73. Visualization studies, using Gfp-labelled derivatives of these strains, showed that AVO110, in contrast to AVO73, colonizes intercellular crevices between neighbouring plant root epidermal cells, a microhabitat of enhanced exudation. Moreover, AVO110, but not AVO73, also colonizes root wounds, described to be preferential penetration sites for R. necatrix infection. This result strongly suggests that AVO110 meets, and can attack, the pathogen on the root. Finally, when co-inoculated with the pathogen, AVO110 utilizes hyphal exudates more efficiently for proliferation than AVO73 does, and colonizes the hyphae more abundantly than AVO73. We conclude that the differences between the strains in colonization levels and strategies are likely to contribute to, and even can explain, the difference in disease-controlling abilities between the strains. This is the first report that shows that two similar bacterial strains, selected by their ability to colonize avocado root, use strongly different root colonization strategies and suggests that in addition to the total bacterial root colonization level, the sites occupied on the root are important for biocontrol.     

Agrobacterium tumefaciens-mediated genetic transformation of the white root rot ascomycete Rosellinia necatrix

Hygromycin B resistance was conferred to the mycelium of the white root rot fungus Rosellinia necatrix by transformation with the hygromycin B phosphotransferase gene (hph) of Escherichia coli under the control of the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. In all three transformants, the presence of hph and single-copy integrations of the marker gene were demonstrated by Southern analysis. This is the first report describing A. tumefaciens-mediated transformation of R. necatrix     

Genetic analysis of barrage line formation during mycelial incompatibility in Rosellinia necatrix

When mycelia of Rosellinia necatrix encounter mycelia of a different genetic strain, distinct barrage lines are formed between the two. These barrages have variable features such as pigmented pseudosclerotia structures, a clear zone, fuzzy hair-like mycelia, or tuft-like mycelia, suggesting that mycelial incompatibility triggers a number of cellular reactions. In this study, to evaluate cellular reactions we performed genetic analysis of mycelial incompatibility of R. nectarix, using 20 single ascospore isolates from single perithecia. Mycelial interaction zones were removed by spatula and cellular reactions studied on oatmeal agar media. The interaction zones were categorized into types such as sharp or wide lines, with or without melanin, and combinations of these. Although various reaction types were observed, we were able to identify a single genetic factor that appears to be responsible for the barrage line formation within oatmeal agar medium. DNA polymorphism analysis identified parental isolates and revealed that R. necatrix has a heterothallic life cycle.     

Armillaria mellea as a cause of oak decline in Hatam-baigh forest of Iran

A200 ha forest of "Hatam-baig" is located in Ardebil Province on the Northwest of Iran. Oak trees (Quercus macranthera Fisch & Mey) in this forest have been faced with declining and extinction since 1991, that has destructed about one third of the forest trees until now. This disorder was expressed in various symptoms including wilting, defoliation and decline. In order to identify factors causing decline, a study was managed from 1998 to 2001. Samples were taken from roots, trunks, crowns and soil beneath the canopy and were cultured on different culture media subsequently. Armillaria mellea (Vahl) P. Kumm., Phytophthora cryptogea Pethybr. & Laff., Dematophora sp., Pythium aphanidermatum (Edson) Fitzp. and Fusarium spp. were the most common isolated fungi. A. mellea appeared to be the essential causal agent of the decline according to the studies made on oak tress decline around the world and based on brown rot observed beneath mycelial fans in the cross section prepared from the trunk and characteristics of the isolated fungi. The fungus activity had been favored by physiological weakness and stresses in oak rootstocks caused by brown- tail moth (Euproctis chrysorhoea L.) and drought stress in infected trees. The biological species of this fungus was identified as Armillaria mellea, using hybridization tests and application of haploid test strains. The fungi such as Phytophthora sp., Pythium sp., and Dematophora sp. can not be infective in this forest due to being hydrophylous. In the southern part of the forest with remarked steepness, the severity of the decline appears to be more than that in the smoothly northern part. The decline of Q. macranthera is reported as matrix nova. The report of the isolated fungi from this oak species is also universally new.     

Suitability of mycorrhiza-defective mutant/wildtype plant pairs (Solanum lycopersicum L. cv Micro-Tom) to address questions in mycorrhizal soil ecology

The ectomycorrhizal (ECM) fungi associated with Pinus thunbergii seedlings grown on sand dune were identified by molecular method, and the diversity of bacteria associated with ECM and Extraradical mycelium were examined by Denaturing Gradient Gel Electrophoresis (DGGE) of PCR-amplified 16S rDNA. The mycorrhizal formation rate of 1-year old P. thunbergii seedlings was more than 95%. Cenococcum geophilum was the most dominant ECM fungus, followed by T01, RFLP-8, Russula spp., and Suillus sp. Bacterial community was most diverse with C. geophilum- and RFLP-8-mycorrhiza. Sequencing analysis showed that Burkholderia spp. and Bradyrhizobium spp. were on the surface of ECM short root of seven ECM. The fungi detected as extraradical mycelium using DGGE of 18S rDNA were Suillus bovinus and RFLP-8-mycorrhiza. Bacterial community on the extraradical mycelium was more diverse than those on ECM root tip. Burkholderia spp. and Bradyrhizobium spp. were found also on extraradical mycelium.     

Diagnosis and management of Sclerotinia stem rot (white mould) of lentils in Greece

The paper describes the field-level symptoms, the identification and management of Sclerotinia stem rot of lentil caused by the soilborne plant pathogen Sclerotinia sclerotiorum in Greece. Regarding symptoms at a field level, initially plants before flowering turn yellow with roots and the base of the plants become brown; then rotten plants exhibit a dry stem and die. On the diseased tissue, at the base of the stem, the typical white mycelium and the resting bodies (sclerotia) were observed. According to our pathogenicity studies in vitro, on the infected plant tissues the fungus first develop a characteristic fluffy white mycelium which will give rise to large black sclerotia, the most obvious evidence of plants infected with S. sclerotiorum. Finally, concerning evaluation of fungicides, isolates of S. sclerotiorum were sensitive to thiophanate-methyl and to triazole fungicides. Thiophanate-methyl and triazole fungicides proved to be most effective in controlling the disease emerged from mycelium or sclerotia.     

Sclerotium cepivorum Berk. in onion (Allium cepa L.) crops: isolation and characterization of bacteria antagonistic to the fungus in Queensland

Bacteria antagonistic to the fungal pathogen Sclerotium cepivorum Berk., which causes onion white rot disease, were isolated and characterized. Eighty percent of the bacterial antagonists isolated from soil samples and sclerotia of S. cepivorum recovered from soil were identified as belonging to the genus Bacillus. The other antagonists were coryneforms and Pseudomonas spp. In contrast, 90% of the antagonists isolated from onion roots were mostly Pseudomonas spp. and the remainder were Erwinia spp. The significance of these antagonistic bacteria is discussed in relation to the control of white rot disease of onion.     

Telomeric fingerprinting of the white root rot fungus,Rosellinia necatrix: a useful tool for strain identification

The telomere associated DNA sequence pTel46, which was isolated from Coprinus cinereus, was hybridized with Rosellinia necatrix genomic DNA. The DNA fragments hybridized with pTel46 were more sensitive to Bal31 nuclease. This result suggests that the DNA fragments hybridized with pTel46 were located at the end of chromosomes in R. necatrix. Telomere-linked restriction fragment length polymorphism (RFLP) was found in strains of R. necatrix isolated from various field and single ascospores. Thus, this marker appears to be an excellent tool to show the great polymorphism of R. necatrix. However, RFLP could not be found among several field isolated strains belonging to the same mycelial compatibility group (MCG) isolated in the same field. Therefore the strains belonging to the same MCG might be the same strain that could be anastomosed with each other without cell death except for strain W718 carrying a double-stranded RNA (dsRNA) virus. Therefore the RFLP corresponded to a MCG group, and none of the strains belonging to the same MCG group showed different RFLP in R. necatrix. Moreover, the presence of a kind of dsRNA virus might imply anastomosis between compatible strains.     

Prolific production of sclerotia in soil by Rhizoctonia solani anastomosis group (AG) 11 pathogenic on lupin

Rhizoctonia solani anastomosis group (AG) 11 causes serious damping‐off and hypocotyl rot of narrow‐leafed lupins (Lupinus angustifolius) in the northern grain‐belt of Western Australia. R. solani AG‐11 produced abundant sclerotia in sand overlaid on potato dextrose agar. Sclerotia were produced in larger numbers in natural Lancelin sand than in Geraldton loamy sand collected from the northern grain‐belt of Western Australia. The majority of the sclerotia produced were in >250 to <500 μam size range. The germination levels of sclerotia in the first two cycles of drying and germination were not significantly different. Sclerotia still retained 50% germination after four such cycles, indicating that they may have the ability to withstand the climatic cycles of the Mediterranean environment of southwestern Western Australia. The radial growth of the mycelium from sclerotia, however, declined with each drying and germination cycle. Inoculum potential of the pathogen increased with the size of sclerotia resulting in more severe lupin hypocotyl rot with larger sclerotia. The number of sclerotia produced in soil increased with increasing density of lupin seedlings. The results also indicate that R. solani AG‐11 can produce sclerotia on infected plant tissues as well as in soil. This is the first report of the prolific production of sclerotia by AG‐11 and their significant role in infection of lupins in soil in Western Australia.     

The survival on chrysanthemum roots of epiphytic mycelium of Mycosphaerella ligulicola

Mycosphaerella ligulicola has been shown to survive as epiphytic mycelium on the root surface of chrysanthemum cuttings: such survival could continue throughout the life of the glasshouse crop. Symptomless surface colonization of roots of cuttings could be induced in non-sterile soil from an inoculum of (a) mycelium and sclerotia or (b) conidia (Ascochyta state); the colonization could spread upwards over the root surface. After 12 weeks survival as an epiphyte on chrysanthemum roots the fungus was still pathogenic to unrooted cuttings. Although the root surfaces of twelve other plants could be colonized by M. ligulicola the fungus survived on these roots for not more than 8 weeks.