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Four isolates of Entoleuca sp., family Xylariaceae, Ascomycota, recovered from avocado rhizosphere in Spain were analyzed for mycoviruses presence. For that, the dsRNAs from the mycelia were extracted and subjected to metagenomics analysis that revealed the presence of eleven viruses putatively belonging to families Partitiviridae, Hypoviridae, Megabirnaviridae, and orders Tymovirales and Bunyavirales, in addition to one ourmia-like virus plus other two unclassified virus species. Moreover, a sequence with 98% nucleotide identity to plant endornavirus Phaseolus vulgaris alphaendornavirus 1 has been identified in the Entoleuca sp. isolates. Concerning the virome composition, the four isolates only differed in the presence of the bunyavirus and the ourmia-like virus, while all other viruses showed common patterns. Specific primers allowed the detection by RT-PCR of these viruses in a collection of Entoleuca sp. and Rosellinia necatrix isolates obtained from roots of avocado trees. Results indicate that intra- and interspecies horizontal virus transmission occur frequently in this pathosystem., This research was funded by the Spanish Ministry of Science and Innovation project AGL2014-52518-C2-2-R and the IFAPA projects AVA201301.13 and AVA201601.14, co-financed by FEDER.

Two monoconidial isolates of Trichoderma atroviride Miller and one of Trichoderma virens Giddens and Foster were combined with the contact fungicide fluazinam to improve the in vitro inhibition of Rosellinia necatrix Prillieux and the control of avocado white root rot caused by this fungus. A fluazinam concentration of 0.01 mg L −1 was obtained as the most effective minimum concentration for in vitro combinations of antagonist-fungicide, significantly reducing (on average, by 41%) the growth of a representative collection of pathogen isolates. The combinations of one isolate of Trichoderma atroviride or virens , plus fluazinam at 0.01 mg L −1 showed a greater in vitro growth inhibition of R. necatrix (36 and 65%, respectively) than the use of fluazinam alone (23%). However, the inhibition of R. necatrix growth by the other T. atroviride plus fluazinam was less than 20% and not significantly different from the control plus fluazinam. The isolate of T. virens applied alone or with fluazinam at 0.01 mg L −1 induced growth inhibition (65%) of the pathogen similar to that induced by the control with fluazinam at a concentration of 0.05 mg L −1 . A fluazinam concentration of 0.01% has been confirmed as effective for controlling white root rot of avocado. Furthermore, the combination of fluazinam at 0.001% with Trichoderma improves significantly the effective control of avocado white root rot on infected plants in comparison with treatments with the fungicide or the antagonist alone. In this way, our results suggest that applications of these antagonist/fungicide combinations should be recommended in order to avoid the development of resistance to the fungicide by the pathogen and improve the control of avocado white root rot.

The improvement in soil quality of avocado crops through organic amendments with composted almond shells has a positive effect on crop yield and plant health, and enhances soil suppressiveness against the phytopathogenic fungus Rosellinia necatrix. In previous studies, induced soil suppressiveness against this pathogen was related to stimulation of Gammaproteobacteria, especially some members of Pseudomonas spp. with biocontrol-related activities. In this work, we isolated bacteria from this suppressiveness-induced amended soil using a selective medium for Pseudomonas-like microorganisms. We characterized the obtained bacterial collection to aid in identification, including metabolic profiles, antagonistic responses, hybridization to biosynthetic genes of antifungal compounds, production of lytic exoenzymatic activities and plant growth-promotion-related traits, and sequenced and compared amplified 16S rDNA genes from representative bacteria. The final selection of representative strains mainly belonged to the genus Pseudomonas, but also included the genera Serratia and Stenotrophomonas. Their biocontrol-related activities were assayed using the experimental avocado model, and results showed that all selected strains protected the avocado roots against R. necatrix. This work confirmed the biocontrol activity of these Gammaproteobacteria-related members against R. necatrix following specific stimulation in a suppressiveness-induced soil after a composted almond shell application.

Fungi are an important component of the soil ecosystem. Mycoviruses have numerous potential impacts on soil fungi, including phytopathogenic fungal species. However, the diversity and ecology of mycoviruses in soil fungi is largely unexplored. Our previous work has shown that the soil-borne phytopathogenic fungus Rosellinia necatrix was infected with several novel mycoviruses after growing for 2-3 years in an apple orchard. In this study, we investigated whether natural infection of R. necatrix with mycoviruses occurs under limited conditions. Virus-free R. necatrix isolates were grown in a small bucket containing soil samples for a short time (1.5-4.5 months) under greenhouse conditions. Screening of dsRNA mycoviruses among 365 retrieved isolates showed that four, including 6-31, 6-33, 6-35, and 7-11, harbored virus-like dsRNAs. Molecular characterization of the dsRNAs revealed that three retrieved isolates, 6-31, 6-33, and 6-35 were infected with a novel endornavirus and isolate 7-11 is infected with a novel partitivirus belonging to the genus Alphapartitivirus. These novel mycoviruses had no overt biological impact on R. necatrix. Overall, this study indicates that natural infections of R. necatrix with new mycoviruses can occur under experimental soil conditions.

The alternaria blackspot of rapeseed is one of the most prominent diseases of rapeseed. It is caused by three species of the genus Alternaria: Alternaria brassicicola, Alternaria brassicae, and Alternaria raphanin. Here we report a novel positive-sense RNA virus from an A. brassicicola strain 817-14. The virus has a 6639 nucleotide (nt) long genome, excluding a poly (A)-tail, and was predicted to contain three putative open reading frames (ORF1, ORF2, and ORF3). The large ORF1 encoded a 174-kDa polyprotein (composed of 1522 amino acid residues) containing a conserved RNA-dependent RNA polymerase (RdRp) domain and a helicase domain. The other two smaller ORFs encoded polypeptides with unknown function. Homology search and phylogenetic analysis, based on the RdRp and helicase domains, suggest that this virus is related to and grouped with Sclerotinia sclerotiorum fusarivirus 1 (SsFV1), Rosellinia necatrix fusarivirus 1 (RnFV1), Fusarium graminearum virus-DK21 (FgV1), and Penicillium roqueforti RNA mycovirus 1 (PrRV1), all of which belong to a newly proposed family Fusariviridae. For this study, we designed the virus as "Alternaria brassicicola fusarivirus 1" (AbFV1). Virus elimination revealed that AbFV1 has no conspicuous impact on the biological properties of its host.

Rosellinia necatrix Prill. is the causal soil-borne agent of avocado white root rot disease. This work proposes a new method of treatment with fungicide to combat this disease, aimed to obtain a quantitative assessment of the evolution of fungal inoculum in soil samples. A total of 39 avocado trees were selected from five commercial orchards affected by R. necatrix. Root samples were collected to recover R. necatrix isolates. The fluazinam treatments were applied around each tree by soil injection, and soil samples were collected before and after each fungicide application to analyze the evolution of the fungus by qPCR. In addition, two samplings of avocado fruits during two harvesting seasons were performed to measure the chemical residues. All 24 R. necatrix isolates taken from avocado roots were pathogenic. The qPCR quantification results showed that the percentage of fungal inoculum in the soil was significantly lower after the first fluazinam treatment compared to that before any fungicide had been applied. Furthermore, soil from non-treated avocado trees did not show any significant differences in the concentration of fungal inoculum. The chemical analysis of the fruits did not show residues. These results provide growers with a new method to control this soil-borne pathogen in crops., This study was partly supported by the Spanish Plan Nacional I+D+I Ministerio de Economía y Competitividad (AGL 2014-52518-C2-2-R). The research was also co-financed by FEDER funds (EU). The authors would like to thank Dr. Fernando Lafont Déniz for his technical assistance on fungicide residual detection, TROPS and PROJOCASA S.A. for their technical support, especially in the location of the orchards, and ISK Biosciences Europe S.A. for providing the fungicide fluazinam needed to carry out this work.

The ascomycete Cryphonectria parasitica has served as a model filamentous fungus for studying virus host interactions because of its susceptibility to diverse viruses, its genetic manipulability and the availability of many biological and molecular tools. Cryphonectria prasitica is known to activate antiviral RNA silencing upon infection by some viruses via transcriptional up-regulation of key RNA silencing genes. Here, utilizing a newly developed GFP-based reporter system to monitor dicer-like 2 (dcl2) transcript levels, we show different levels of antiviral RNA silencing activation by different viruses. Some viruses such as mycoreovirus 1, a suppressor-lacking mutant of Cryphonectria hypovirus 1 (CHV1-Δp69) and Rosellinia necatrix partitivirus 11 (RnPV11) highly induced RNA silencing, while others such as CHV3, Rosellinia necatrix victorivirus 1 and RnPV19 did not. There was considerable variation in dcl2 induction by different members within the family Hypoviridae with positive-sense single-stranded RNA genomes or Partitiviridae with double-stranded RNA genomes. Northern blotting and an in vitro Dicer assay developed recently by us using mycelial homogenates validated the reporter assay results for several representative virus strains. Taken together, this study represents a development in the monitoring of Dicer activity in virus-infected C. parasitica.

The white root rot fungus, Rosellinia necatrix , damages a wide range of fruit trees. R. necatrix is known to host a variety of mycoviruses, and several of these have potential as biological control agents. RNA interference (RNAi) is a fungal defense mechanism against viral infection, and it is therefore important to understand the RNAi amplification and transmission systems in R. necatrix for effective use of mycoviruses in disease control. In this study, we describe an intriguing RNAi signal transmission phenomenon in R. necatrix . In R. necatrix transformants with autonomously replicating vectors carrying a hairpin structure to induce RNAi, the gene silencing effect was distributed locally and unevenly, based on the vector distribution. This indicates that R. necatrix has no mechanism to propagate silencing signals systemically, unlike Caenorhabditis elegans and Arabidopsis thaliana . Furthermore, the expression of RNA-dependent RNA polymerase homologs was not upregulated during RNAi induction, suggesting that silencing signals are not amplified at sufficient levels to induce systemic RNAi in R. necatrix . Our results also suggest that, in addition to hairpin-induced RNAi, there is either a 5′ transitive RNAi or quelling-like gene silencing system in R. necatrix . This is the first study demonstrating that systemic RNAi is not induced by local RNAi in fungi.

Avocado white root rot is a disease caused by the soil-borne pathogenic ascomycete Rosellinia necatrix Prill. In this work, we have carried out different experiments combining the contact fungicide fluazinam with biocontrol rhizobacterial strains of Pseudomonas chlororaphis and Bacillus subtilis, in order to reduce in vitro growth inhibition of R. necatrix and to control the disease in avocado plants. When combining fluazinam at a low concentration (0.01 mg L−1) with individual biocontrol bacterial strains, a slight reduction in fungal growth was observed (10 mm2 for PCL1606; 19 mm2 for PCL1601 and 11 mm2 for PCL1608) when compared with the use of the fungicide alone (31 mm2). From all the assayed rhizobacteria, the combined use of the rhizobacterial strain Pseudomonas chlororaphis PCL1606 with fluazinam showed the best fungal inhibition in in vitro tests (22–66%). The protective effect against avocado root rot in avocado plants was recorded using a concentration of fluazinam of 0.01%, but its effect was not improved when combined this fungicide with the biocontrol rhizobacteria PCL1606. However, when a lower concentration of fluazinam was tested (0.001%), the combination with PCL1601 or PCL1606 strains resulted in improved control of the disease than when using the fungicide alone with a reduction of 15% and 8% respectively. These results point to the development of an integrated tool to control avocado white root rot by using lower concentrations of fungicide, combined with a suitable biocontrol rhizobacteria, thus obtaining the protective effect on the plant, but reducing the chemical residues and preventing the development of fungal resistance., This study was supported partly by the Plan Nacional I + D + I Ministerio de Economía y Competitividad (AGL2014-52518-C2-1-R and AGL2014-52518-C2-2-R) Spain. In addition, this research was co-financed by FEDER funds (EU).

Avocado white root rot (WRR) is a disease of significant importance in the Mediterranean area and is caused by the emerging pathogen Rosellinia necatrix. Five Pseudomonas spp. and three Bacillus subtilis strains were selected for this study based on their rhizospheric soil survival, antagonistic and biocontrol activities under experimental conditions. After a single inoculation, Pseudomonas spp. showed higher persistence in rhizospheric soil, and B. subtilis strains were not detected for longer than ten days. In biocontrol assays, B. subtilis CB115 protected the plants against WRR under experimental growth chamber conditions at a similar level to the biocontrol reference strain Pseudomonas chlororaphis PCL1606. The strains B. subtilis CB115, P. chlororaphis CB254 and PCL1606 and Pseudomonas fluorescens CB306 were further selected to explore their biocontrol abilities under commercial greenhouse conditions. B. subtilis CB115 showed consistent biocontrol with protection levels similar to the biocontrol strain P. chlororaphis PCL1606. Although B. subtilis CB115 did not show persistence features, repeated applications by direct soil drenching led to the detection of a stable population that was mainly composed of spores in the avocado rhizospheric soil after 74 days. Our results indicate that both P. chlororaphis PCL1606 and B. subtilis CB115 can help control R. necatrix under commercial greenhouse conditions. This is the first study investigating the performance of biocontrol bacteria against avocado WRR under commercial greenhouse settings.

Here we report the biological and molecular attributes of a novel dsRNA virus isolated from Rosellinia necatrix, a filamentous phytopathogenic fungus. The virus, termed Rosellinia necatrix quadrivirus 1 (RnQV1), forms rigid spherical particles approximately 45nm in diameter in infected mycelia. The particles contain 4 dsRNA segments, dsRNA1 to dsRNA4, with a size range of 4.9 to 3.7kbp, each possessing a single large ORF. A comparison of the virus-infected and -cured isogenic fungal strains suggested that RnQV1 infection has no appreciable phenotypic effects. Phylogenetic analysis using the dsRNA3-encoded RdRp sequence revealed that RnQV1 is more distantly related to quadripartite chrysoviruses than to monopartite totiviruses, and is placed in a distinct group from other mycoviruses. No significant sequence similarities were evident between known proteins and RnQV1 structural proteins shown to be encoded by dsRNA2 or dsRNA4. These suggest that RnQV1 is a novel latent virus, belonging to a new family.

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.

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.

Five isolates of Trichoderma atroviride and one isolate each of T. virens , T. harzianum and T. cerinum were tested for in vivo biological control of white root rot of avocado ( Rosellinia necatrix ). Five of these Trichoderma isolates were previously selected as possible biological control agents on the basis of their capacity to control the disease and high levels of colonization of the avocado rhizoplane. Combinations of the five selected isolates were evaluated on cellophane for compatibility with each other and T. virens CH 303 was eliminated because of a high incompatibility with other Trichoderma isolates. The four remaining isolates, all T. atroviride , were tested singly and in combination for their capacity to control avocado white root rot. Isolate CH 304.1 provided the highest levels of control when tested singly or in combination with isolate CH 101.

Benomyl, carbendazim, thiophanate methyl and fluazinam were tested in vitro and in vivo for their effects on Rosellinia necatrix. Benomyl, carbendazim and thiophanate methyl at ⩾0.5 μg ml−1 totally inhibited R. necatrix mycelial growth on PDA medium. At 0.1 μg ml−1, carbendazim and fluazinam inhibited growth by 97% and 84%, respectively, in comparison with fungicide-free medium. Benomyl and thiophanate methyl had less effect (53% and 22%, respectively) at this dose. Mycelia treated with fungicide were tested for their capacity to grow when transferred to fungicide-free medium one month after treatment, demonstrating the fungistatic effect of fluazinam in comparison with the other systemic fungicides assayed. The in vivo effects of fungicides on white root rot (WRR) control in avocado plants was studied in two greenhouse experiments with eight sequential applications of fungicides at doses of 0.1% (w/v) and 1% (w/v). Plants were inoculated once with R. necatrix or inoculated twice when plants received an additional dose of inoculum 30 days after the last application of fungicide. Fluazinam was highly effective in the control of WRR as measured by aerial symptoms, plant height increase, dry weight of secondary roots, percentage isolation of R. necatrix from secondary roots and R. necatrix populations in soil. Carbendazim, benomyl and thiophanate methyl were less effective.

Biological control of soil-borne pathogens is frequently based on the application of antagonistic microorganisms selected solely for their ability to produce in vitro antifungal factors. The aim of this work was to select bacteria that efficiently colonize the roots of avocado plants and display antagonism towards Rosellinia necatrix, the causal agent of avocado white root rot. A high frequency of antagonistic strains (ten isolates, 24.4%) was obtained using a novel procedure based on the selection of competitive avocado root tip colonizers. Amplification and sequencing of the 16S rRNA gene, in combination with biochemical characterization, showed that eight and two of the selected isolates belonged to the genera Pseudomonas and Stenotrophomonas, respectively. Characterization of antifungal compounds produced by the antagonistic strains showed variable production of exoenzymes and HCN. Only one of these strains, Pseudomonas sp. AVO94, produced a compound that could be related to antifungal antibiotics. All of the ten selected strains showed twitching motility, a cell movement involved in competitive colonization of root tips. Production of N-acyl-homoserine lactones and indole-3-acetic acid was also reported for some of these isolates. Resistance to several bacterial antibiotics was tested, and three strains showing resistance to only one of them were selected for biocontrol assays. The three selected strains persisted in the rhizosphere of avocado plants at levels considered crucial for efficient biocontrol, 10(5)-10(6) colony forming units/g of root; two of them, Pseudomonas putida AVO102 and Pseudomonas pseudoalcaligenes AVO110, demonstrated significant protection of avocado plants against white root rot.

Rosellinia species have been recorded all over the world. Many species occur as saprobes and only a few are well-known root pathogens. Although Rosellinia spp. have been recognized for more than 130 years to cause losses in many economically important crops and trees, the few existing sets of quantitative data have not previously been collated. Our objective is to integrate available knowledge on the biology and control of Rosellinia root rots, with emphasis on the tropics and subtropics, i.e. root rots caused by Rosellinia bunodes , R. necatrix and R. pepo. The review will summarize briefly the taxonomy, geographical distribution, ecology, host-range, disease symptoms and spread of Rosellinia in order to provide a rational basis for the choice of control options. Different avenues explored to control Rosellinia are presented: cultural control, solarization, genetic resistance, chemical and biological control. Successful Rosellinia control usually consists of an integrated approach, invariably centring around cultural control options. In the short term, chemical control may be best suited to supplement cultural measures, while medium and long term solutions could be provided by biological control and genetic resistance, respectively, if prioritized research needs are addressed satisfactorily.

Dematophora necatrix causes a tuber rot of cultivated Cyperus esculentus in eastern Spain. The disease is spread through infected tubers. To overcome this problem, the use of a hot-water treatment (HWT) of C. esculentus tubers was studied. Isolates of D. necatrix from C. esculentus showed sensitivity to temperatures above 34°C, indicating HWT could be used as a practical way of destroying tuber-borne inoculum of this pathogen. Temperatures from 43°C to 64°C for three periods of time (10, 20 or 30 min) were applied to healthy tubers. These tubers tolerated temperatures of 55°C from 10 to 30 min without a reduction in sprouting. Recovery of D. necatrix was not possible by isolations on potato dextrose agar +500 mg l−1 of streptomycin sulfate from internal fragments of affected tubers treated at 55°C for 10, 20 or 30 min. Two field trials, applying a HWT at 53°C for 25 min and 55°C for 25 min, respectively, on infected planting material, showed normal plant development without reduction in yield and a good control of the disease. HWT at 53–55°C for 25–30 min has been recommended to control tuber-borne inoculum.

Many small (temporary) collections of fungi maintained by plant pathologists during their research receive inadequate attention to ensure stability. Maintaining collections of fungi in pure and viable conditions, minimising physiological and morphological changes is, however, a necessity. The objective of this study was to find preservation techniques for three Rosellinia isolates used in our plant pathogenic research. Various inert and nutritious carriers, solid as well as liquid, were used to test their suitability for conserving these Rosellinia isolates. Different cryoprotectants, cooling rates and thawing rates were tested to optimise liquid nitrogen storage procedures. Survival and/or growth rate were assessed over time. Rosellinia bunodes was the most difficult to store with survival not exceeding six to nine months using traditional storage methods in mineral oil and silica gel. Storage of Rosellinia necatrix and Rosellinia pepo was successful for periods up to at least 16 months in several carriers and for up to two years for R. necatrix in silica gel. Storage in liquid nitrogen proved no problem for R. necatrix or R. pepo with a 100% survival in all cases, although radial growth rates after recuperation were affected by cryoprotectant and thawing rates. Storage of R. bunodes was more difficult and survival as well as growth rates were affected by cryoprotectant and thawing rates. Cooling rates did not affect radial growth in any of the isolates. The results showed that development of a generalised procedure for storage of our Rosellinia species was not possible and successful storage protocols had to be developed for individual isolates.

The fermentation product of herbs by lactic acid bacteria (FHL) was assayed for antifungal activities against Rosellinia necatrix, Helicobasidium mompa, Fusarium oxysporum, Pythium graminicola and Pyricularia oryzae. FHL completely inhibited the growth of R. necatrix, H. mompa, P. graminicola and P. oryzae, and reduced the growth of F. oxysporum by 35%. When the seeds of Medicago sativa L. (alfalfa), Asparagus officinalis L. (asparagus), Brassica campestris L. (komatsuna), Oryza sativa L. (rice), Spinacia oleracea L. (spinach), Festuca arundinacea Schreb. (tall fescue), and Lycopersicum esculentum Mill. (tomato) were put on plates containing 0.69 mg/ml FHL, their germination rates did not decrease. The root elongation of A. officinalis, B. campestris, O. sativa, and L. esculentum seedlings was suppressed on plates containing 6.92 mg/ml FHL, but the root elongation of M. sativa was not suppressed on the 6.92 mg/ml FHL plate. When FHL was diluted to less than 1.73 mg/ml, the diluted FHL solution did not suppress the germination of B. campestris seeds, but it suppressed the root elongation of B. campestris seedlings. An FHL concentration higher than 0.35 mg/ml hastened the growth of seedlings of B. campestris in the presence of a chemical fertilizer but delayed the growth of these seedlings in the absence of the chemical fertilizer, suggesting that inorganic elements could affect the efficiency of FHL.

A somatic incompatibility system was demonstrated in Rosellinia necatrix (anamorph Dematophora necatrix) through barrage formation in pairings among mass fungal isolates from infected avocado trees in commercial orchards in southern Spain, as well as among single ascospore isolates derived from perithecia formed in infected roots. Results revealed the occurrence of high diversity in somatic incompatibility in the R. necatrix population in the study. The occurrence of R. necatrix perithecia in naturally infected avocado roots in the field suggests that such a diversity might originate from sexual reproduction. However, the possibility that other parasexual mechanisms demonstrated in this fungus may contribute to the genetic diversity can not be discounted.

The effects of pre- and post-planting soil solarization on white root rot of apple caused by Rosellinia necatrix were examined in two naturally infested orchards. The pathogen was eradicated in solarized soil to a depth of 30 cm. Partial or complete destruction of the pathogen was obtained in solarized-shaded (partially shaded by the tree canopy) plots. No reinfestation of solarized and solarized-shaded soil was observed 2 years after treatment. No death of replanted apple trees occurred in the solarized plots up to 2 years after solarization, whereas 60% of trees died in untreated plots. No disease developed during the third year after solarization in an existing apple orchard, but a low rate of disease recurred in the fourth year. In contrast, mortality in diseased trees in the absence of solarization reached 100%, 4 years after treatment. Two previously solarized soils, out of seven tested, caused reduced growth of the pathogen, indicating induction of soil suppressiveness by solarization.