Olpidium Bornovanus Research Articles

On the TPPP Protein of the Enigmatic Fungus, Olpidium-Correlation between the Incidence of p25alpha Domain and That of the Eukaryotic Flagellum.

Loss of the flagellum was an important step in the evolution of fungi. The flagellated fungi of the phylum Olpidiomycota are the closest relative of the non-flagellated terrestrial fungi. There are genes encoding proteins, the occurrence of which shows a strong correlation with the incidence of the flagellum. One of these gene/protein families is "TPPP-like proteins" whose main feature is the presence of the p25alpha domain. The functional link between TPPP and flagellum has also been shown. Most of the phyla of flagellated fungi have been known to contain TPPP-like proteins but Olpidiomycota was an exception. This study demonstrates that Olpidium bornovanus, similarly to some fungi of Chytridiomycota and Blastocladiomycota, has a "fungal-type" TPPP characterized by the presence of two (a complete and an incomplete) p25alpha domains.

Diversity of soil-borne fungal species associated to root rot and vine decline of melon in Sardinia (Italy)

Monosporascus root rot and vine decline of melon (MRRVD) is a destructive disease complex mainly occurring in semiarid cultivation areas. In the last decade, in the melon producing area of Sardinia (Italy), yield reductions up to 100% were recorded due to the occurrence of MRRVD. The present study aimed to undertake a two-year survey of fungal pathogens associated with MRRVD grown as a monocrop in two locations in Central Sardinia, Sinis, and Sassu, and to investigate the possible role of soil fungal diversity in the disease development. Melon plants were affected by similar symptoms but colonized by a set of soil-borne fungal pathogens different between surveyed sites and cropping seasons, including Plectosphaerella melonis, P. cucumerina, Fusarium solani, Macrophomina phaseolina and Monosporascus cannonballus. Olpidium bornovanus and O. virulentus were isolated using bait plants and detected by NGS analysis. Bait plants had a general decrease in biomass and yellowing of foliage. The presence of most of the isolated pathogens was also confirmed by the NGS analysis of the soil microbiome. Our results confirm that among the fungal complex implicated in the occurrence of RRVD in Sardinia, O. bornovanus along with O. virulentus likely assume a key role in the development of the disease alone and possibly in association with other pathogens. Differences in the pathogenic fungal spectrum here recorded in symptomatic roots may be associated with agricultural practices, soil physicochemical characteristics, and fungal community composition and function profile.

Genome-scale phylogenetic analyses confirm Olpidium as the closest living zoosporic fungus to the non-flagellated, terrestrial fungi

The zoosporic obligate endoparasites, Olpidium, hold a pivotal position to the reconstruction of the flagellum loss in fungi, one of the key morphological transitions associated with the colonization of land by the early fungi. We generated genome and transcriptome data from non-axenic zoospores of Olpidium bornovanus and used a metagenome approach to extract phylogenetically informative fungal markers. Our phylogenetic reconstruction strongly supported Olpidium as the closest zoosporic relative of the non-flagellated terrestrial fungi. Super-alignment analyses resolved Olpidium as sister to the non-flagellated terrestrial fungi, whereas a super-tree approach recovered different placements of Olpidium, but without strong support. Further investigations detected little conflicting signal among the sampled markers but revealed a potential polytomy in early fungal evolution associated with the branching order among Olpidium, Zoopagomycota and Mucoromycota. The branches defining the evolutionary relationships of these lineages were characterized by short branch lengths and low phylogenetic content and received equivocal support for alternative phylogenetic hypotheses from individual markers. These nodes were marked by important morphological innovations, including the transition to hyphal growth and the loss of flagellum, which enabled early fungi to explore new niches and resulted in rapid and temporally concurrent Precambrian diversifications of the ancestors of several phyla of fungi.

Genome Analysis of Melon Necrotic Spot Virus Incursions and Seed Interceptions in Australia

Melon necrotic spot virus (MNSV) was detected in field-grown Cucumis melo (rockmelon) and Citrullus lanatus (watermelon) plants in the Sunraysia district of New South Wales and Victoria, Australia, in 2012, 2013, and 2016, and in two watermelon seed lots tested at the Australian border in 2016. High-throughput sequencing was used to generate near full-length genomes of six isolates detected during the incursions and seed testing. Phylogenetic analysis of the genomes suggests that there have been at least two incursions of MNSV into Australia and none of the field isolates were the same as the isolates detected in seeds. The analysis indicated that one watermelon field sample (L10), the Victorian rockmelon field sample, and two seed interception samples may have European origins. The results showed that two isolates (L8 and L9) from watermelon were divergent from the type MNSV strain (MNSV-GA, D12536.2) and had 99% nucleotide identity to two MNSV isolates from human stool collected in the United States (KY124135.1, KY124136.1). These isolates also had high nucleotide pairwise identity (96%) to a partial sequence from a Spanish MNSV isolate (KT962848.1). The analysis supported the identification of three previously described MNSV genotype groups: EU-LA, Japan melon, and Japan watermelon. To account for the greater diversity of hosts and geographic regions of the MNSV isolates used in this study, it is suggested that the genotype groups EU-LA, Japan melon, and Japan watermelon be renamed to groups I, II, and III, respectively. The divergent isolates L8 and L9 from this study and the stool isolates from the United States formed a fourth genotype group, group IV. Soil collected from the site of the Victorian rockmelon MNSV outbreak was found to contain viable MNSV and the virus vector, a chytrid fungus, Olpidium bornovanus (Sahtiyanci) Karling, 18 months after the initial MNSV detection. This is a first report of O. bornovanus from soil sampled from an MNSV-contaminated site in Australia.

Brazilian fungal diversity represented by DNA markers generated over 20years.

Molecular techniques using fungal DNA barcoding (ITS) and other markers have been key to identifying the biodiversity of different geographic areas, mainly in megadiverse countries. Here, we provide an overview of the fungal diversity in Brazil based on DNA markers of phylogenetic importance generated since 1996. We retrieved fungal sequences of ITS, LSU, SSU, tef1-α, β-tubulin, rpb1, rpb2, actin, chitin synthase, and ATP6 from GenBank using different field keywords that indicated their origin in Brazil. A total of 19,440 sequences were recovered. ITS is the most representative marker (11,209 sequences), with 70.1% belonging to Ascomycota, 18.6% Basidiomycota, 10.2% unidentified, 1.1% Mucoromycota, two sequences of Olpidium bornovanus (Fungi incertae sedis), one sequence of Blastocladiomycota (Allomyces arbusculus), and one sequence of Chytridiomycota (Batrachochytrium dendrobatidis). Considering the sequences of all selected markers, only the phyla Cryptomycota and Entorrhizomycota were not represented. Based on ITS, using a cutoff of 98%, all sequences comprise 3047 OTUs, with the majority being Ascomycota (2088 OTUs) and Basidiomycota (681 OTUs). Previous numbers based mainly on morphological and bibliographical data revealed 5264 fungal species from Brazil, with a predominance of Basidiomycota (2741 spp.) and Ascomycota (1881 spp.). The unidentified ITS sequences not assigned to a higher taxonomic level represent 1.61% of all ITS sequences sampled and correspond to 38 unknown class-level lineages (75% cutoff). A maximum likelihood phylogeny based on LSU illustrates the fungal classes occurring in Brazil.

First report of Olpidium bornovanus and O. virulentus on watermelon in Sardinia, Italy

First report of Olpidium bornovanus and O. virulentus on watermelon in Sardinia, Italy

Involvement of Olpidium bornovanus and O. virulentus in the occurrence of melon root rot and vine decline caused by Monosporascus cannonballus in central Italy

Monosporascus root rot and vine decline (MRRVD), caused by Monosporascus cannonballus , has become one of the most important diseases of melon worldwide. Recent evidences suggest that M. cannonballus is not the sole cause of MRRVD, but other pathogens, Olpidium spp. and Melon necrotic spot virus (MNSV) are also involved. The aim of this study was to ascertain the potential concomitant involvement of these pathogens in the development MRRVD of melon in Central Italy analyzing a soil sampled from a greenhouse with a known history of this disease. O. bornovanus and O. virulentus along with M. cannonballus were identified from the sampled soil. Bait melon plants grown in the contaminated soil were colonized by both Olpidium species and by M. cannonballus, showing roots browning and foliage wilt. No infection by MNSV was detected in bait plants. In the tripartite pathogenicity test, O. bornovanus alone was found to be a virulent pathogen, capable of colonizing roots with a high percentage of colonization intensity, resulting in root browning and foliage wilting. Both disease severity and intensity of Olpidium colonization determined by the co-inoculation of ascospores of M. cannonballus and Olpidium spp. were statistically similar to that resulted by Olpidium or by ascospores separately inoculated. Root symptoms were accompanied by a gradual vine decline and foliage wilting. Root rot and vine decline, which was previously attributed in Central Italy primarily to M. cannonballus, needs to be reattributed, since O. bornovanus and O. virulentus are also involved without apparent synergistic effect among the pathogens.

Low pH soil inhibits transmission of <i>Melon necrotic spot virus</i> by its fungal vector <i>Olpidium bornovanus </i>

Low pH soil inhibits transmission of <i>Melon necrotic spot virus</i> by its fungal vector <i>Olpidium bornovanus </i>

First Report of Olpidium bornovanus and O. virulentus on Melon in Italy.

A survey for the presence of Olpidium spp. on melon (Cucumis melo L.) was conducted during the beginning of 2013 in central Italy in an unheated greenhouse, located in the melon-producing coastal area of north Latium (central Italy, Viterbo Province) (42°23'09.31″N, 11°30'46.10″E) with a history of monosporascus root rot and vine decline (MRRVD). For this aim, 10 soil samples were collected adjacent to the roots of plants symptomatic of MRRVD, represented by root lesions and rots and loss of smaller feeder roots. Olpidium was baited from collected infested soil by growing melon (cv. Dinero) plants for 45 days. Bait plants grown in sterilized soil were used as negative controls. All the baited melon roots were analyzed by morphological and molecular methods. For the morphological analysis, feeder roots were clarified in a 1.5% KOH solution for 24 h (2) and observed under a light microscope to record the presence or absence of sporangia and resting spores of Olpidium spp., which were observed in baited melon plants grown in infested soil and not in control roots. In particular, stellate resting spores were referred to as O. virulentus because this species cannot be distinguished from O. brassicae, which does not colonize melon. O. bornovanus had smooth-walled resting spores with a honeycomb-like pattern (2). For molecular analysis, DNA was extracted from 21 melon roots and tested by multiplex PCR to confirm Olpidium spp. identification (2). Based on molecular identification, O. virulentus was identified in 40% of samples, and O. bornovanus was identified in 10%. There were no mixed infections in the same sample. Two amplified PCR products, corresponding to O. bornovanus and O. virulentus expected fragment sizes of 977 and 579 bp respectively, were sequenced (GenBank Accession Nos. KF661295 and KF661296). BLAST analysis of the sequences showed 99% nucleotide identity with O. bornovanus isolate CH from Japan collected in melon roots (AB205215) and O. virulentus isolate HY-1 from Japan collected in lettuce roots as reported by Sasaya and Koganezawa (3) (AB205204, formerly O. brassicae). At the end of the experiment, the root systems of all inoculated plants appeared brown, whereas neither symptoms nor sporangia and resting spores were observed in roots of control plants. Olpidium spp. are root-infecting plant pathogens of melon (4), acting as vectors of Melon necrotic spot virus (MNSV) and other destructive plant viruses (1). Moreover, they are directly involved in the induction of germination of ascospores of Monosporascus cannonballus, the causal agent of MRRVD of cucurbits (4). To our knowledge, this is the first report of O. virulentus and O. bornovanus on melon in Italy.

First Report of Vine Decline of Mature Watermelon Plants Caused by Olpidium bornovanus.

In late May 2013, collapse of mature watermelon plants (Citrullus lanatus L.) at first harvest occurred in several drip-irrigated commercial fields in the Coachella Valley, California. Above-ground symptoms consisted of chlorosis, wilting, and death of leaves starting at the crown and progressing rapidly towards the tip of vines. Structural roots of collapsed plants appeared healthy but feeder roots exhibited a brownish discoloration. Microscopic examination revealed that almost all epidermal cells of feeder roots contained either sporangia or resting spores of a fungus tentatively identified, based upon morphological characteristics, as Olpidium bornovanus (Sahtiy.) Karling. No other fungi or fungal-like organisms were microscopically observed in or isolated from structural roots, feeder roots, or vascular tissue of collapsed plants. Leaf, root, and peduncle samples from collapsed plants were tested for Melon necrotic spot virus (MNSV), a virus known to be transmitted by O. bornovanus, and Squash vein yellowing virus (SqVYV), a whitefly-transmitted ipomovirus known to cause watermelon vine decline (1). No MNSV was detected using previously described methods (3). No SqVYV was detected by testing total RNA from symptomatic plants (RNeasy Plant Mini Kit, Qiagen, Valencia, CA) with reverse transcription-PCR using previously described primers and methods (1,2). Genomic DNA was extracted from zoospores of the fungus which were obtained from a single-sporangial isolate maintained on watermelon seedlings. Analysis of ITS 1 and 2 gene sequences and a subsequent search in NCBI GenBank revealed a 99% identity to nucleotide sequences for O. bornovanus (Accession Nos. AB205215 and AB665758). To confirm Koch's postulates, roots of three 5-day-old watermelon seedlings were inoculated by exposure to zoospores (~1 × 105) in a beaker for 2 min and then transplanted into pots containing vermiculite. Pots were irrigated daily and incubated in a growth chamber (25°C, 12-h photoperiod). Controls consisted of non-inoculated watermelon seedlings. The experiment was repeated twice. Within 15 days of inoculation, all inoculated plants were stunted, and roots of stunted plants were brown and most root epidermal cells were filled with either sporangia or resting spores of O. bornovanus. Within 30 days of inoculation, 40 to 60% of the inoculated plants died in all three experiments. No other microorganisms were microscopically observed in or isolated from necrotic roots. Control plants remained symptomless over the duration of the study. Although O. bornovanus has been reported as a root pathogen of melons in greenhouse conditions (3), this is the first worldwide report of the fungus as a root pathogen of watermelons and its association with a late season vine decline in the field. Near-saturated soil conditions resulting from a daily irrigation regime during the latter part of the growing season apparently favored extensive root colonization by this indigenous and opportunistic zoosporic fungus, suggesting that growers should exercise care regarding the duration and frequency of irrigation events.

Effect of soil matric water potentials on germination of ascospores of Monosporascus cannonballus and colonization of melon roots by zoospores of Olpidium bornovanus

Results document, for the first time, the role of soil moisture on a unique, tripartite, host-specific rhizosphere interaction (i.e., Cucumis melo-Monosporascus cannonballus-Olpidium bornovanus). Specifically, colonization of cantaloupe roots by zoospores of O. bornovanus and germination of ascospores of M. cannonballus were highest at a soil matric potential of −0.001 MPa but significantly inhibited at a matric potential of only −0.01 MPa. Matric water potentials of −0.01 MPa or drier are characteristically inhibitory to the motility of zoosporic microbes but not hyphal growth of filamentous fungi like M. cannonballus. These results support our previous conclusion that germination of ascospores of M. cannonballus, a destructive root pathogen of cantaloupe is mediated by O. bornovanus, an obligate, zoosporic fungus.

メロンえそ斑点ウイルスを媒介する<i>Olpidium bornovanus </i>土壌汚染程度の段階的検出法

メロンえそ斑点ウイルスを媒介するOlpidium bornovanus の土壌中の動態を把握することは，メロンえそ斑点病の被害回避を図る際に重要である。そこで，nested PCR を用い，被検土壌の本菌汚染程度を段階的に検出する検出法の開発を試みた。被検土壌から抽出したDNA を鋳型とする直接検出法では，O. bornovanus 汚染土壌からnested PCR のsecondary reaction でのみ菌が検出され，段階的な検出はできなかった。一方，被検土壌を宿主植物であるメロンへ添加後，１または２週間栽培した宿主根から抽出したDNA を鋳型とする間接検出法では，菌の培養期間とnested PCR の段階の組み合わせで検出限界が異なり，この検出限界の差に基づき，菌の汚染程度が段階的に検出できる可能性が示唆された。更に，簡易DNA 抽出法や検出能の高いポリメラーゼの利用により作業や検出日数短縮等の省力化が可能であった。

INFLUENCE OF GRAFTED WATERMELON PLANT DENSITY ON YIELD AND QUALITY IN SOIL INFESTED WITH MELON NECROTIC SPOT VIRUS

Abstract The decrease in fruit yield and quality caused by soil diseases is one of the major problems of watermelon production. Soil disinfection with methyl bromide (MB) has been used to prevent fungus attacks; however, its use is being restricted because this substance damages the ozone layer. Field experiments were carried out in open field in soils infested with Olpidium bornovanus and Melon necrotic spot virus (MNSV) in Colima, Mexico, where watermelons had only been grown previously using soil fumigation with MB. Yield and quality of the watermelon cultivar ‘Triploid Tri-X 313’ grafted on two rootstocks of Cucurbita maxima × Cucurbita moschata (‘RS841’ and ‘Shintosa Camelforce’) were evaluated during 2007-2008. Each experiment had five treatments, two of them with non-grafted plants at a density of 3472 plants/ha, with and without MB fumigation. The remaining three treatments had grafted plants in non-fumigated soil with plant densities of 2778, 2083, and 1736 plants/ha. The use of watermelon grafted on ‘RS841’ and ‘Shintosa Camelforce’ rootstocks significantly increased the average fruit weight and the total yield of watermelon. Grafting may be considered as an alternative to MB fumigation. With the use of grafted watermelon plants, planting density may be reduced by 50%, obtaining higher yields than those obtained from non-grafted plants grown in fumigated soil. Fruits harvested from grafted plants had higher firmness than those harvested from non-grafted plants, without affecting the content of soluble solids. INTRODUCTION

Comparisons of ribosomal DNA-internal transcribed spacer sequences and biological features among Olpidium bornovanus isolates from Cucurbitaceae-cultivating soil in Japan

The chytrid fungus Olpidium bornovanus is an obligate plant parasite that acts as a vector to transmit Melon necrotic spot virus in cultivated soil. Here, we conducted a molecular phylogenetic analysis of 16 isolates of O. bornovanus taken from soil in which Cucurbitaceae plants had been grown in various locations throughout Japan. The ribosomal DNA and internal transcribed spacer region sequences of the 16 O. bornovanus isolates were divided into four molecular phylogenetic groups, designated O.bor-A to O.bor-D. Biological features of O. bornovanus isolates such as host specificity varied consistently with their molecular phylogenetic groups.

Olpidium bornovanus-Mediated Germination of Ascospores of Monosporascus cannonballus: A Host-Specific Rhizosphere Interaction

Monosporascus cannonballus, a host-specific root-infecting ascomycete, is the causal agent of a destructive disease of melon (Cucumis melo L.) known as vine decline. Ascospores germinate only in the rhizosphere of melon plants growing in field soil. However, no germination occurs in the rhizosphere of melon plants if the field soil is heated to temperatures >50°C prior to infestation with ascospores. This observation suggested that germination is mediated by one or more heat-sensitive members of the soil microflora. Although bacteria or actinomycetes were heretofore suspected as the germination-inducing microbes, our data demonstrate that Olpidium bornovanus, an obligate, host-specific, root-infecting zoosporic fungus, is responsible. In four experiments conducted in autoclaved field soil amended with various population densities of culturally produced ascospores, significant ascospore germination was recorded only in the rhizosphere of cantaloupe seedlings colonized by O. bornovanus.

Planting density for grafted melon as an alternative to methyl bromide use in Mexico

One of the major challenges of melon ( Cucumis melo L.) yield is the decrease of fruit yield and quality caused by soil diseases. Soil disinfection with methyl bromide (MB) has been used to prevent soil-borne diseases damage; however, use of MB is being restricted because this substance damages the ozone layer. Searching for new MB alternatives, field experiments were carried out in open fields in soils infested with Fusarium oxysporum f. sp. melonis, Olpidium bornovanus, and Melon Necrotic Spot Virus (MNSV) in Colima, Mexico, where melons had only been grown previously using soil fumigation with MB. Yield and quality of melon cultivar Cruiser F1 (Harris Moran seed company) grafted on two rootstocks of Cucurbita maxima Duchesne × Cucurbita moschata Duchesne (“RS841”; Séminis seed company and “Shintosa Camelforce”; Nunhens seed company) were evaluated during two consecutive years. Each experiment had five treatments, two of which with non-grafted plants at a density of 18,519 plants/ha, with and without MB fumigation. The other three treatments consisted of grafted plants in non-fumigated soil with plant densities of 14,815, 11,111, and 9260 plants/ha. The use of melon grafted on “RS841” and “Shintosa Camelforce” rootstocks significantly increased the average fruit weight and the total yield of melons in soil without the need for MB. Grafting may be considered an alternative to MB fumigation. With the use of grafted melon plants, planting density may be reduced by 60%, obtaining higher yields than those obtained from non-grafted plants grown on fumigated land. Fruits harvested from grafted plants had equal firmness than those harvested from non-grafted plants, without affecting the content of soluble solids.

The protruding domain of the coat protein of Melon necrotic spot virus is involved in compatibility with and transmission by the fungal vector Olpidium bornovanus

The Chi and W strains of Melon necrotic spot virus (MNSV) are efficiently transmitted by isolates Y1 and NW1, respectively, of the fungal vector Olpidium bornovanus. Analysis of chimeric viruses constructed by switching the coat protein (CP) gene between the two strains unveiled the involvement of the CP in the attachment of MNSV to zoospores of a compatible isolate of O. bornovanus and in the fungal transmission of the virus. Furthermore, analysis of the chimeric virus based on the Chi strain with the protruding domain of the CP from strain W suggested the involvement of the domain in compatibility with zoospore. Comparison of the three-dimensional structures between the CP of the two MNSV strains showed that many of the differences in these amino acid residues are present on the surface of the virus particles, suggesting that these affects the recognition of fungal vectors by the virus.

Genetic diversity of Melon necrotic spot virus and Olpidium isolates from different origins

The geographic incidence, genetic diversity and phylogenetic relationships of Melon necrotic spot virus (MNSV) and Olpidium isolates were studied in three cucurbit species from several Latin American and European countries on different collecting dates. Of the 112 cucurbit samples analysed, 69 from Guatemala, Honduras, Mexico, Panama and Spain were DAS‐ELISA‐positive for MNSV. Olpidium bornovanus and O. virulentus infections, and MNSV infections mixed with these Olpidium species, were observed for all these countries. Twenty‐nine MNSV isolates from all the origins where the virus was detected were selected and amplified by RT‐PCR. The resulting RT‐PCR of the p29, p89, p7A, p7B and p42 proteins was used to estimate the genetic diversity and the phylogenetic relationships of the MNSV population. The sequences obtained in this study were compared with the MNSV sequences of the NCBI database, and three groups were recovered by nucleotide composition according to geographical origins: the EU‐LA genotype group (with two subgroups: EU and LA, European and Latin American isolates, respectively), the JP melon genotype group (Japanese melon reference isolates) and the JP watermelon genotype group (Japanese watermelon reference isolates). The genetic diversity in the entire p7A and p7B proteins of MNSV suggests that these coding regions are under strong selective pressure. Additionally, the rDNA‐ITS region was analysed in 40 O. bornovanus and O. virulentus isolates associated with each geographical location and host examined. Phylogenetic analysis showed two groups for each Olpidium species, and these groupings were related to the host from which they were originally isolated.

Pathogenicity and Management of Olpidium bornovanus, a Root Pathogen of Melons.

Greenhouse studies document, for the first time, that Olpidium bornovanus, an obligate, holocarpic, root-inhabiting zoosporic fungus heretofore regarded as a nonpathogenic parasite, is a root pathogen. Significant browning of the roots and reductions in shoot and root growth were recorded within 28 days following inoculation of melons with the fungus. Amending the recirculating nutrient solution with either a nonionic surfactant (Agral 90) or a strobilurin fungicide (azoxystrobin) resulted in efficacious management of the disease caused by the fungus.

Efficacy of composting infected plant residues in reducing the viability of Pepper mild mottle virus, Melon necrotic spot virus and its vector, the soil-borne fungus Olpidium bornovanus

Survival of Pepper mild mottle virus (PMMV) and Melon necrotic spot virus (MNSV) in diseased plant residues of pepper and melon, respectively, was studied in several experiments investigating drying and composting conducted in experimental heaps. The viability of the soil-borne fungus Olpidium bornovanus, the vector of MNSV, in infected melon root residues was also evaluated. Maximum temperatures in the heap reached 61.9–73.8 °C during composting processes. Drying of plant residues reduced the infectivity of MNSV whereas the infectivity of PMMV and O. bornovanus was unaffected by drying. Neither the virus nor the fungus was viable after composting. PMMV antigens where detected by serology after 30 and 60 days composting in the four experiments, but virus infectivity was detected only in one process after 30 days. Our results show that the use of composts using plant residues from PMMV and MNSV-infected crops as organic amendments in SE Spain do not appear to pose a risk of re-introducing pathogens to soil. Environmental conditions in that area are suitable for the adequate elimination of these pathogens from infected vegetable residues through composting even during winter.