A Reovirus Causes Hypovirulence of Rosellinia necatrix

ABSTRACT White root rot, caused by Rosellinia necatrix, is a serious soilborne disease of fruit trees and other woody plants. R. necatrix isolate W370 contains 12 segments of double-stranded RNA (dsRNA) that is believed to represent a possible member of the family Reoviridae. W370 was weakly virulent and its hyphal-tip strains became dsRNA free and strongly virulent. The 12 segments of W370dsRNA were transmitted to hygromycin B-resistant strain RT37-1, derived from a dsRNA-free strain of W370 in all or none fashion through hyphal contact with W370. The W370dsRNA-transmitted strains were less virulent than their parent strain RT37-1 on apple seedlings, with mortality ranging between 0 to 16.7% in apple seedlings that were inoculated with the W370dsRNA-containing strains and 50 to 100% for seedlings inoculated with the dsRNA-free strains. Some W370dsRNA-containing strains killed greater than 16.7% of seedlings, but these were found to have lost the dsRNA in planta. These results indicate that W370dsRNA is a hypovirulence factor in R. necatrix. In addition, a strain lost one segment (S8) of W370dsRNA during subculture, and the S8-deficient mutant strain also exhibits hypovirulence in R. necatrix.     

Analysis of population structure of Rosellinia necatrix on Cyperus esculentus by mycelial compatibility and inter-simple sequence repeats (ISSR)

Genetic diversity was studied in a population of 24 isolates of Rosellinia necatrix obtained from Cyperus esculentus and 16 from other hosts by means of mycelial compatibility groups (MCGs) and inter-simple sequence repeat (ISSR) techniques. All isolates obtained from C. esculentus belonged to a unique MCG, irrespective of their geographical origin or year of isolation, and were incompatible with the isolates from other hosts. ISSR analysis gave results which were in agreement with the MCG determination; selected ISSR primers grouped a subset of eight representative isolates from C. esculentus in a unique cluster. The homogeneity found within the population of R. necatrix from C. esculentus in Valencia province could suggest that the pathogen was introduced recently and has spread further via infected tubers inadvertently used as propagating material, as well as by cull tubers swept away by irrigation water.     

Metabolic responses of avocado plants to stress induced by Rosellinia necatrix analysed by fluorescence and thermal imaging

European Journal of Plant Pathology - One of the most important soilborne diseases affecting avocado (Persea americana Mill.) crops is white root rot, caused by the fungus Rosellinia necatrix. In...     

Comparison of conventional and molecular methods for the detection of Rosellinia necatrix in avocado orchards in southern Spain

White root rot, caused by Rosellinia necatrix , is one of the most important diseases in avocado orchards and is particularly widespread on the Mediterranean seaboard of southern Spain. In this study, the presence of the pathogen in soil samples collected from the base of 47 plants showing different symptoms of canopy decline was assessed with a molecular detection method based on real-time Scorpion PCR. Results were compared with symptoms in the canopy and with the traditional method of isolation of R. necatrix from roots and/or bark. The fungus was isolated from 24 samples by the traditional method and from 37 soil samples by the molecular method (cycle threshold values 25·8 to 47·1), demonstrating the higher sensitivity and reliability of the molecular method. A single real-time PCR amplification was sufficient to detect R. necatrix in naturally infested soils. The avoidance of nested PCR has important practical implications because of the reduced costs and risk of cross contamination. Also, it enables faster sample analysis and is more appropriate for quantitative detection. A modified molecular method was also developed to detect R. necatrix in roots and in soils with very low populations of the pathogen.     

Control of Rosollinia necatrix by Deep Placement and Hot Treatment with Methyl Bromide1

Rosellinia necatrix , the cause of white root rot in fruit trees, was efficiently controlled in an infested avocado orchard soil, by applying methyl bromide by deep injection (up to 90 cm) of cold gas and by surface application of hot gas, at a dosage (for heavy soil) of 1500 kg/ha and 1000 kg/ha, respectively. Efficiency of control was evaluated: 1) by testing pieces of naturally diseased avocado roots buried in the soil before treatment at different depths up to 100 cm; 2) by planting avocado and almonds in the treated field and in soil samples; 3) by burying avocado leaves in the soil samples. All the evaluaton tests showed that both treatments of methyl bromide were effective in controlling R. necatrix completely up to the depth of 100 cm, immediately and 9 months after treatment.     

Inoculation of Lupinus luteus with White Root Rot Fungus, Rosellinia necatrix, to Estimate Virulence

An inoculation method using Lupinus luteus was developed for estimating virulence of isolates of the white root rot fungus, Rosellinia necatrix Prillieux. Fungal cultures grown on pieces of mulberry twigs were placed in contact with the hypocotyl of 3-week-old seedlings growing in pots of soil. Disease development was uniform and reproducible in repeated experiments. Of 24 isolates with double-stranded RNA, eight were weakly virulent. This method is useful throughout the year for estimating virulence of many isolates of the fungus and for screening for hypovirulent isolates. Received 2 March 2001/ Accepted in revised form 25 May 2001     

我国甘薯脱毒种薯种苗繁育存在的问题及建议

病毒病是甘薯的重要病害, 种植脱毒健康种苗是防治病毒病?提高甘薯产量最有效的方法?近年来, 我国甘薯病毒及其传播介体的发生呈现出新的特点, 传统的脱毒种薯种苗繁育体系不能满足当前甘薯生产的需要?本文综述了当前我国甘薯病毒的种类及危害现状, 分析了我国甘薯脱毒种薯种苗繁育中存在的问题, 对规范和完善我国甘薯脱毒种薯种苗繁育体系提出了建议?     

梨果生刺盘孢子囊孢子单孢菌系的生物学特性及致病性研究

 梨果生刺盘孢的2种致病型菌株FJ-85(产生黑点症状)和菌株FJ-11-2(产生坏死斑症状)的子囊孢子单孢培养均可产生正、负2种类型的单孢菌系。正型单孢菌系菌落浅白色,其上形成的子囊孢子再培养,可形成正、负2种类型的单孢菌系;负型单孢菌系菌落深灰色,其上形成的子囊孢子再培养,只形成负型一种单孢菌系。2个菌株的单孢菌系中,负型的比例明显多于正型。2种类型的单孢菌系对峙培养,在菌落内及菌落交界处均可形成子囊壳,而同种类型的单孢菌系对峙培养仅在菌落内产生子囊壳。结果表明同种类型的单孢菌系可同宗配合,而正、负2种类型的单孢菌系还可异宗配合。正型单孢菌系的致病力与原始菌株相近,而负型单孢菌系较原始菌株弱。2个菌株的单孢菌系在翠冠梨上引起的症状差异与其原始菌株相同。研究结果为解析梨果生刺盘孢的有性生殖与致病的关系提供了新的有用信息。     

板栗疫病菌脱毒方法的比较研究

 病毒与宿主相互作用中,存在宿主主动或被动摆脱病毒的机制。本研究用光照诱导-单孢分离、原生质体再生和菌丝尖端分离3种方法对携带低毒病毒的板栗疫病菌(Cryphonectria parasitica)的EP721、EP713和Euro7三个菌株的脱毒效率进行了比较。结果表明:经光照诱导-单孢分离后所有菌株均可获得脱毒菌株,而原生质体再生对EP721的脱毒效率最高,是继光照诱导-单孢分离脱毒法之后另一种新的有效的脱毒方法,特别适用于不产孢的真菌脱毒。     

Antimicrobial Activity of Culture Filtrate of Bacillus amyloliquefaciens RC-2 Isolated from Mulberry Leaves

ABSTRACT A potential antagonist, Bacillus amyloliquefaciens strain RC-2, against Colletotrichum dematium, mulberry anthracnose fungus, was obtained from healthy mulberry leaves by in vitro and in vivo screening techniques. Application of culture filtrate of RC-2 inhibited disease on mulberry leaves, indicating that suppression was due to antifungal compounds in the filtrate. Development of mulberry anthracnose on mulberry leaves was inhibited only when the culture filtrate was applied before fungal inoculation, and it was not inhibited by application after inoculation. These results suggest that the antifungal compounds in the filtrate exhibit a preventive effect on the disease. Peptone significantly increased production of the antifungal compounds. The culture filtrate of RC-2 also inhibited the growth of several other phytopathogenic fungi and bacteria, such as Rosellinia necatrix, Pyricularia oryzae, Agrobacterium tumefaciens, and Xanthomonas campestris pv. campestris, in vitro. From the culture filtrate of RC-2, seven kinds of antifungal compounds were isolated by high performance liquid chromatography analysis, and one of the compounds was determined as iturin A2, a cyclic peptide, by nuclear magnetic resonance and fast atom bombardment mass analysis.     

Biological control of avocado white root rot with combined applications of Trichoderma spp. and rhizobacteria

This study tested the effectiveness of single and combined applications of Trichoderma and rhizobacterial strains to control white root rot (WRR) caused by Rosellinia necatrix in avocado plants. Three Trichoderma, two T. atroviride and one T. virens monoconidal strains and four bacterial strains (Bacillus subtilis, Pseudomonas pseudoalcaligenes and two P. chlororaphis) were assayed to determine their compatibilities in vitro. In addition, the effects of the bacterial filtrates were evaluated against the Trichoderma strains and reciprocally; these filtrates were applied alone or in combination to determine their effectiveness against R. necatrix. Individual control agents or combinations of them were applied to avocado plants that were artificially inoculated with a virulent R. necatrix strain. Compatibility between the combined Trichoderma applications and the bacterial strains was observed and these combinations significantly improved the control of R. necatrix during the in vitro experiments. A relative protective effect of some Trichoderma and bacteria was observed on the control of avocado WRR when they were applied singly. The combinations of T. atroviride strains with bacterial strains P. chlororaphis and P. pseudoalcaligenes showed a better control of avocado WRR, whereas the rest of Trichoderma and bacteria combinations also reduced significantly the level of disease and induced a delay at the onset of disease with respect to avocado plants inoculated either with Trichoderma or bacteria.     

The pathogenicity of Vairimorpha necatrix (Microspora: Microsporidia) against the tomato moth, Lacanobia oleracea (Lepidoptera: Noctuidae) and its potential use for the control of lepidopteran glasshouse pests

A droplet feeding technique was used to feed known amounts of Vairimorpha necatrix (Kramer) spores to larvae of the tomato moth, Lacanobia oleracea (L) in order to assess the susceptibility of this lepidopteran pest to the pathogen. All first- to fourth-instar larvae died as a result of ingesting 1000 or more V necatrix spores. Two forms of death were observed, which were dependent on the dose and the age of the insect when treated. For first-instar larvae, rapid death (within 6days of dosing) occurred after ingestion of 2000 spores, whereas lower doses resulted in a proportion of larvae dying from chronic infection (microsporidiosis). For more advanced stages, increasing spore doses were required to give rapid death, such that a dose of 200,000 spores was needed to give 80% mortality within 6 days for third-instar larvae. Rapid death was not observed in fourth- to sixth-instar larvae. In all cases successful pupation and adult emergence were much reduced compared with non-infected larvae. Suspensions of V necatrix were sprayed on to tomato (Lycopersicon esculentum Mill) plants maintained in small glasshouses prior to infestation of the plants with L oleracea larvae. The numbers and biomass of pest larvae retrieved from the plants sprayed with V necatrix were significantly reduced by up to 40% and 70%, respectively, compared with plants sprayed with water (control). Similarly, plants sprayed with V necatrix showed a reduction in damage of up to 45% compared with the control plants.     

Exogenous application of melatonin improves eradication of apple stem grooving virus from the infected in vitro shoots by shoot tip culture

Production and maintenance of virus-free planting materials is pivotal for the control of viral diseases. The present study attempted to test exogenous application of melatonin for eradication of apple stem grooving virus (ASGV) from virus-infected in vitro shoots of apple cultivar Gala. Exogenous application of 15 μm melatonin to the shoot proliferation medium significantly increased the number of shoots and shoot length. The level of endogenous indole-3-acetic acid (IAA) was the highest in the shoots proliferating on the shoot proliferation medium containing 15 μm melatonin. Shoot regrowth levels were significantly higher in shoot tips of the virus-infected shoots cultured for 4 weeks on this medium than the control. In addition, culture of shoot tips of the virus-infected in vitro shoots proliferated for 4 weeks on this medium resulted in 95% of shoots being virus-free, while no virus-free shoots were obtained in shoot tips of the virus-infected shoots cultured without melatonin. Analyses by microtissue direct RT-PCR and RT-qPCR showed that ASGV concentration decreased in shoot tips of the virus-infected shoots proliferating on the medium containing 15 μm melatonin for 4 weeks. Virus localization showed that exogenous application of melatonin enlarged the virus-free area in the virus-infected shoot tips. These data provide explanations as to why exogenous application of melatonin can efficiently eradicate ASGV. Exogenous application of melatonin provides an alternative means for plant virus eradication and has the potential to produce virus-free plants.     

Characteristics of strains of Septoria nodorum adapted to wheat or to barley

Sixty-four strains of Septoria which had been isolated from a range of hosts at different locations and in different years were characterized for their adaptation to wheat or barley, growth at near-maximum temperature, fluorescence, colony morphology, conidial (pycnidiospore) length and hexokinase and alkaline phosphatase isozymes. For each character except conidial length, the strains could be divided into two or three discrete groups. The variation in these six characters was strongly associated, such that 60 strains could be classified into two groups, designated W-type and B-type. W-type strains are adapted to wheat, produce large colonies at 31 C, fluoresce, produce brown-pigmented colonies, and have fast isozymes. B-type strains are adapted to barley, produce small colonies at 31 C, do not fluoresce, produce pink-pigmented colonies, and have slow isozymes. A few strains differed from these norms in one of the six characters, but only one showed an atypical host adaptation. The four unclassified strains differed from W- or B-type in two or more characters. The many differences between the W- and B-types suggest they are genetically distinct populations within Septoria nodorum.     

Loss of Viability of Dematophora necatrix in Solarized Soils

To study the relationship between temperature regimes and loss of viability of Dematophora necatrix in soil, two field experiments were conducted to determine the effectiveness of soil solarization on reducing the population of D. necatrix colonizing avocado root segments buried at a depth of 15–60cm. Increase of maximum hourly temperatures attributable to soil solarization reached, depending on depth, 6.7–4.6°C in unshaded areas and 3.9–1.5°C for shaded areas in the first experiment (starting in early June, 1995). The better environmental conditions in the second experiment (starting by mid-July, 1995) led to higher temperature increases (8.6–5.6°C, depending on depth) when solarization was conducted in unshaded areas. One, 4, 5 and 6 weeks of solarization were required to eliminate the viability of D. necatrix at 15, 30, 45 and 60cm depths in the first experiment, whereas only 8, 10, 15 and 22 days of solarization were needed for the loss of viability of D. necatrix at the same depths in the second experiment. In shaded areas, however, soil solarization attained significant effectiveness at 15cm depth.Regression analyses of fungal viability (ln-transformed data) over accumulated temperature–time showed best fits when the minimum threshold temperature was 30°C. Although eradication of D. necatrix in soil can be achieved down to 60cm depth in solarized plots, and at 15cm depth in unsolarized unshaded plots, the accumulation of temperature–time appeared less effective in reducing inoculum viability in the latter.     

Vegetative incompatibility in Diaporthe ambigua

Vegetative compatibility of strains of Diaporthe ambigua has not previously been examined. Single ascospore and single ascus strains, originating from individual apple, pear and plum rootstocks, were paired on freshly prepared oatmeal agar to determine if vegetative incompatibility could be detected in D. ambigua . Barrage reactions were evident as black lines along the zone of mycelial contact between expanding colonies (vegetative incompatibility reaction). Strains from cankers within an area were of numerous vegetative compatibility groups (VCGs). Strains from adjacent rootstocks usually differed in VCG. D. ambigua has the ability to outcross, and does so, despite its apparent homothallic nature. DsRNA-containing strains of D. ambigua developed a broad, clear zone when paired with a dsRNA-containing strain from a different VCG.     

Measuring the sterol content of barley leaves infected with powdery mildew as a means of assessing partial resistance to Erysiphe graminis f.sp. hordei

Barley leaf segments of several partially resistant cultivars were inoculated with different strains and combinations of strains of Erysiphe graminis. Infection level was assessed by counting colony number, by measuring sporulating area using an image analyser, and by extracting and quantifying sterols. Good correlations were obtained between data from all three methods. One cultivar contained less sterol than expected from the number of colonies present but some of the resistance in that cultivar was known to be due to reduced colony size. Image analysis was unable to discern the reduced colony size component of resistance. Scanning electron microscopy revealed that single colonies may originate from more than one conidial infection. Sterol analysis was the most representative method of comparing the reaction of diverse host genotypes to mildew infection.     

Movement of aphid-transmitted Sugarcane yellow leaf virus (ScYLV) within and between sugarcane plants

Sugarcane yellow leaf virus (ScYLV) is distributed worldwide and has been shown to be the cause of the disease sugarcane yellow leaf syndrome (YLS). This study was an investigation of the transmission and spread of ScYLV in Hawaii. Several aphids are known to transmit the virus, but investigation of infestation and transmission efficiency showed Melanaphis sacchari to be the only vector important for field spread of the disease. The initial multiplication of ScYLV in a virus-free plant occurred exclusively in very young sink tissues. When a single leaf was inoculated on a plant, that leaf and all older leaves remained virus-free, based on tissue-blot immunoassay, whereas meristems and all subsequently formed new leaves became infected. Therefore, only after those leaves which had already developed before inoculation had been shed, did the complete plant contain ScYLV. Spread of the viral infection to neighbouring plants in the plantation fields via aphids was relatively slow and in the range of a few metres per year. No indication of long-distance transfer could be seen. This indicates that it may be possible to produce and use virus-free seed cane for planting of high-yielding but YLS-susceptible cultivars.     

Combining proteolytic and phloroglucinol-producing bacteria for improved biocontrol of Pythium-mediated damping-off of sugar beet

Pseudomonas fluorescens F113 and Stenotrophomonas maltophilia W81 protect sugar beet from Pythium -mediated damping-off through production of the antifungal secondary metabolite 2,4-diacetylphloroglucinol and extracellular proteolytic activity, respectively. In this study, the two biocontrol strains were combined in a consortium, with the objective of improving upon the level of protection achieved when using each strain singly. Growth and in vitro production of 2,4-diacetylphloroglucinol by F113 and extracellular lytic enzymes by W81 were not affected when inoculated in combination. The abilities of W81 and F113 to colonize the rhizosphere of sugar beet were essentially similar when the two strains were applied singly or coinoculated onto seeds in a 1 : 1 ratio, both in natural soil microcosms and under field conditions. Concomitantly, single inoculation with W81 or F113 effectively prevented colonization of sugar beet seeds by Pythium spp. in soil microcosms, without the necessity for combining both strains. However, this parity was not reflected in seed emergence where the combination of W81 and F113 significantly enhanced final sugar beet stands (to the level achieved with chemical pesticides) under microcosm conditions at 28 days after sowing. In a field experiment, the only inoculation treatment capable of conferring effective protection of sugar beet was that in which W81 and F113 were coinoculated, and this treatment proved equivalent to the use of chemical fungicides. In conclusion, when compared with single inoculations of either biocontrol strain, the combined use of a phloroglucinol-producing P. fluorescens and a proteolytic S. maltophilia improved protection of sugar beet against Pythium -mediated damping-off.