Cytological analysis of mycelial incompatibility in Rosellinia necatrix

When the mycelia of Rosellinia necatrix encounter mycelia with a different genetic background, distinct barrage lines form. In this study, we observed hyphal interactions between compatible and incompatible R. necatrix pairs by means of light and electron microscopy. Although we observed perfect hyphal anastomosis in compatible pairs of isolates, the hyphae never anastomosed in incompatible pairs (i.e., the hyphae remained parallel or crossed over without merging). These behaviours appeared to result from the detection of or failure to detect one or more diffusible factors. The attraction to other hyphae in pairs of incompatible isolates was increased by supplementation of the growing medium with activated charcoal, although no anastomosis was observed and ultrastructural observation confirmed a lack of hyphal anastomosis. Programmed cell death (PCD) started with one of the two approaching hyphae. Heterochromatin condensation and genomic DNA fragmentation were not observed. Moreover, cell damage began with the tonoplast and continued with the plasma and nuclear membranes, suggesting that the PCD observed in heterogenic incompatibility of R. necatrix was a vacuole-mediated process.     

On the independence of barrage formation and heterokaryon incompatibility in Neurospora crassa

A barrage is a line or zone of demarcation that may develop at the interface where genetically different fungi meet. Barrage formation represents a type of nonself recognition that has often been attributed to the heterokaryon incompatibility system, which limits the co-occurrence of genetically different nuclei in the same cytoplasm during the asexual phase of the life cycle. While the genetic basis of the heterokaryon incompatibility system is well characterized in Neurospora crassa, barrage formation has not been thoroughly investigated. In addition to the previously described Standard Mating Reaction barrage, we identified at least three types of barrage in N. crassa; dark line, clear zone, and raised aggregate of hyphae. Barrage formation in N. crassa was evident only when paired mycelia were genetically different and only when confrontations were carried out on low nutrient growth media. Barrages were observed to occur in some cases between strains that were identical at all major heterokaryon incompatibility (het) loci and the mating-type locus, mat, which acts as a heterokaryon incompatibility locus during the vegetative phase of N. crassa. We also found examples where barrages did not form between strains that had genetic differences at het-6, het-c, and/or mat. Taken together, these results suggest that the genetic control of barrage formation in N. crassa can operate independently from that of heterokaryon incompatibility and mating type. Surprisingly, barrages were not observed to form when wild-collected strains of N. crassa were paired. However, an increase in the frequency of pairings that produced barrages was observed among strains obtained by back-crossing wild strains to laboratory strains, or through successive rounds of inbreeding of wild-derived strains, suggesting the presence in wild strains of genes that suppress barrage.     

Cytological analysis of mycelial incompatibility in Helicobasidium mompa

When the mycelia of Helicobasidium mompa encounter mycelia with a different genetic background, distinct demarcation lines form. The hyphae of H. mompa induce heterogenic incompatibility accompanied by active programmed cell death (PCD) process. In this study, we observed hyphal interaction between compatible and incompatible H. mompa pairs by means of light and electron microscopy. PCD started with one of the two approaching hyphae. Heterochromatin condensation and genomic DNA laddering were not observed. Moreover, cell damage began with the tonoplast and continued with the plasma membrane and nuclear membrane, suggesting that the PCD observed in heterogenic incompatibility of H. mompa is a vacuole-mediated process.     

Studies on the Metabolic Products of Rosellinia necatrix Berlese

A new inhibitor of plant growth, named rosellinic acid, C₁₁H₁₀O₅, m.p. 206~208°C, has been isolated from the culture filtrate of Rosellinia necatrix Berlese. Rosellinic acid has one C-methyl, one phenolic hydroxyl and one aryl carboxylic acid group. Fusion with alkali gave 4,5-dihydroxyisophthalic acid, and the results of physical and chemical tests on the rosellinic acid led to the conclusion that the formula of 6-carboxy-8-hydroxy-2-methylchromanone seems the most possible structure.     

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.     

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.     

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     

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.     

GFP sheds light on the infection process of avocado roots by Rosellinia necatrix

In order to monitor Rosellinia necatrix infection of avocado roots, we generated a plasmid vector (pCPXHY1eGFP) constitutively expressing EGFP and developed a protoplast transformation protocol. Using this protocol, four R. necatrix isolates were efficiently transformed and were shown to stably express EGFP homogeneously while not having any observable effect on pathogenicity. Confocal laser scanning microscopy (CLSM) images of avocado roots infected with the highly virulent isolate CH53-GFP demonstrated that fungal penetration of avocado roots occurs simultaneously at several random sites, but it occurs preferentially in the crown region as well as throughout the lenticels and in the junctions between epidermal cells. Not only were R. necatrix hyphae observed invading the epidermal and cortical root cells, but they were also able to penetrate the primary and secondary xylem. Scanning electron microscopy (SEM) images allowed detailed visualisation of the hyphal network generated by invasion of R. necatrix through the epidermal, cortical and vascular cells, including hyphal anastomosis and branching points. To our knowledge, this is the first report describing the construction of GFP-tagged strains belonging to the genus Rosellinia for monitoring white root rot using CLSM and SEM.     

Observations on the teleomorph of the white root rot fungus,Rosellinia necatrix, and a related fungus,Rosellinia aquila

The process of teleomorph development in the white root rot fungusRosellinia necatrix is described on diseased roots of Japanese pear. Stromata were also found on dead plants in nonagricultural lands such as yards and forests. The stroma ofR. aquila is also described. Research based on the program for Promotion of Basic Research Activities for Innovative Biosciences.     

Potentiation of Mycovirus Transmission by Zinc Compounds via Attenuation of Heterogenic Incompatibility in Rosellinia necatrix

Heterogenic incompatibility is considered a defense mechanism against deleterious intruders such as mycovirus. Rosellinia necatrix shows strong heterogenic incompatibility. In the heterogenic incompatibility reaction, the approaching hyphae hardly anastomosed, a distinctive barrage line formed, and green fluorescent protein (GFP)-labeled hyphae quickly lost their fluorescence when encountering incompatible hyphae. In this study, transmission of a hypovirulence-conferring mycovirus to strains with different genetic backgrounds was attempted. Various chemical reagents considered to affect the programmed cell death pathway or cell wall modification were examined. Treatment with zinc compounds was shown to aid in transmission of mycoviruses to strains with different genetic backgrounds. In incompatible pairings, treatment with zinc compounds accelerated hyphal anastomosis; moreover, cytosolic GFP was transmitted to the newly joined hyphae. These results suggest that zinc compounds not only increase hyphal anastomosis but also attenuate heterogenic incompatibility.     

Genetic analysis of interspecific incompatibility in Brassica rapa

In interspecific pollination of Brassica rapa stigmas with Brassica oleracea pollen grains, pollen tubes cannot penetrate stigma tissues. This trait, called interspecific incompatibility, is similar to self-incompatibility in pollen tube behaviors of rejected pollen grains. Since some B. rapa lines have no interspecific incompatibility, genetic analysis of interspecific incompatibility was performed using two F₂ populations. Analysis with an F₂ population between an interspecific-incompatible line and a self-compatible cultivar ‘Yellow sarson’ having non-functional alleles of S-locus genes and MLPK, the stigmas of which are compatible with B. oleracea pollen grains, revealed no involvement of the S locus and MLPK in the difference of their interspecific incompatibility phenotypes. In QTL analysis of the strength of interspecific incompatibility, three peaks of LOD scores were found, but their LOD scores were as high as the threshold value, and the variance explained by each QTL was small. QTL analysis using another F₂ population derived from selected parents having the highest and lowest levels of interspecific incompatibility revealed five QTLs with high LOD scores, which did not correspond to those found in the former population. The QTL having the highest LOD score was found in linkage group A02. The effect of this QTL on interspecific incompatibility was confirmed by analyzing backcrossed progeny. Based on synteny of this QTL region with Arabidopsis thaliana chromosome 5, a possible candidate gene, which might be involved in interspecific incompatibility, is discussed.     

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.     

Presence and distribution of double-stranded RNA elements in the white root rot fungus Rosellinia necatrix

Double-stranded (ds)RNA of various types was detected in 65 (21.8%) of 298 isolates from vegetative hyphae of Rosellinia necatrix by electrophoresis, but dsRNA was not detected from 39 ascosporic isolates. There were 45 distinct dsRNA profiles in the 65 isolates: they varied in the number of electrophoretic bands from 1 to 12 and in size from less than 1000 bp to more than 10 kbp. Each dsRNA profile was unique to each locality. dsRNAs having the same profiles were restricted to isolates of the same mycelial compatibility groups (MCG) from the same trees, with an exception where different profiles were detected in different isolates of the same MCGs. Received: May 7, 2001 / Accepted: September 5, 2001     

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...     

Developing tools to unravel the biological secrets of Rosellinia necatrix, an emergent threat to woody crops

White root rot caused by Rosellinia necatrix is one of the most destructive diseases of many woody plants in the temperate regions of the world, particularly in Europe and Asia. Recent outbreaks of R. necatrix around the globe have increased the interest in this pathogen. Although the ecology of the disease has been poorly studied, recent genetic and molecular advances have opened the way for future detailed studies of this fungus. TAXONOMY: Rosellinia necatrix Prilleux. Kingdom Fungi; subdivision Ascomycotina; class Euascomycetes; subclass Pyrenomycetes; order Sphaeriales, syn. Xylariales; family Xylariaceae; genus Rosellinia. IDENTIFICATION: Fungal mycelium is present on root surfaces and under the bark, forming mycelium fans, strands or cords. A typical presence of pear-shaped or pyriform swellings can be found above the hyphal septum (with diameters of up to 13 μm). Sclerotia are black, hard and spherical nodules, several millimetres in diameter. Black sclerotia crusts may also form on roots. On synthetic media, it forms microsclerotia: irregular rough bodies composed of a compact mass of melanized, interwoven hyphae with no differentiated cells. Chlamydospores are almost spherical (15 μm in diameter). Synnemata, also named coremia (0.5-1.5 mm in length), can be formed from sclerotia or from mycelial masses. Conidia (3-5 μm in length and 2.5-3 μm in width) are very difficult to germinate in vitro. Ascospores are monostichous, situated inside a cylindrical, long-stalked ascus. They are ellipsoidal and cymbiform (36-46 μm in length and 5.5-6.3 μm in width). HOST RANGE: This fungus can attack above 170 different plant hosts from 63 genera and 30 different families, including vascular plants and algae. Some are of significant economic importance, such as Coffea spp., Malus spp., Olea europaea L., Persea americana Mill., Prunus spp. and Vitis vinifera L. DISEASE SYMPTOMS: Rosellinia necatrix causes white (or Dematophora) root rot, which, by aerial symptoms, shows a progressive weakening of the plant, accompanied by a decline in vigour. The leaves wilt and dry, and the tree can eventually die. White cottony mycelium and mycelial strands can be observed in the crown and on the root surface. On woody plant roots, the fungus can be located between the bark and the wood, developing typical mycelium fans, invading the whole root and causing general rotting. DISEASE CONTROL: Some approaches have been attempted involving the use of tolerant plants and physical control (solarization). Chemical control in the field and biological control methods are still under development.     

A moderate level of hypovirulence conferred by a hypovirus in the avocado white root rot fungus,Rosellinia necatrix

Two isolates of Rosellinia necatrix (Rn118-8 and Rn480) have previously obtained from diseased avocado trees in commercial orchards of the coastal area in southern Spain. Rn118-8 and Rn480 have weak virulence on avocado plants, and are infected by R. necatrix hypovirus 2 (RnHV2). In this work, the possible biological effects of the hypovirus on R. necatrix were tested. First, RnHV2 was transmitted from each of Rn118-8 and Rn480 to a highly virulent, RnHV2-free isolate of R. necatrix (Rn400) through hyphal anastomosis, using zinc compounds which attenuate the mycelial incompatibility reactions and allow for horizontal virus transfer between vegetatively incompatible fungal strains. Next, we carried out an analysis of growth rate in vitro and a virulence test of these newly infected strains in avocado plants. We obtained five strains of Rn400 infected by RnHV2 after horizontal transmission, and showed some of them to have lower colony growth in vitro and lower virulence on avocado plants compared with virus-free Rn400. These results suggest that R. necatrix isolates infected by RnHV2 could be used as novel virocontrol agents to combat avocado white root rot.     

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.     

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.