Pathogen emergence in changing ecosystems: RAD-seq reveals long evolutionary history of Phellinus noxius in east Asia, Australia, and the Pacific Islands

Emerging pathogens have been increasing exponentially over the last century. The knowledge on whether these organisms are native to ecosystems or have been recently introduced is often of great importance. Understanding the ecological and evolutionary processes promoting emergence can help to control their spread and forecast epidemics. Using restriction site-associated DNA sequencing data, we studied genetic relationships, pathways of spread, and evolutionary history of Phellinus noxius, an emerging root-rotting fungus of unknown origin, in eastern Asia, Australia, and the Pacific Islands. We analyzed patterns of genetic variation using Bayesian inference, maximum likelihood phylogeny, populations splits and mixtures measuring correlations in allele frequencies and genetic drift, and finally applied coalescent based theory using approximate Bayesian computation (ABC) with supervised machine learning. Population structure analyses revealed five genetic groups with signatures of complex recent and ancient migration histories. The most probable scenario of ancient pathogen spread is movement from west to east: from Malaysia to the Pacific Islands, with subsequent spread to Taiwan and Australia. Furthermore, ABC analyses indicate that P. noxius spread occurred thousands of generations ago, contradicting previous assumptions that it was recently introduced in multiple areas. Our results suggest that recent emergence of P. noxius in east Asia, Australia, and the Pacific Islands is likely driven by anthropogenic and natural disturbances, including deforestation, land-use change, severe weather events, and introduction of exotic plants. This study provides a novel example of utilization of genome wide allele frequency data to unravel dynamics of pathogen emergence under conditions of changing ecosystems.

PATOGENISITAS ISOLAT Phellinus noxius PADA JAMBU METE DAN BEBERAPA JENIS TANAMAN BERKAYU LAINNYA

<p>Gejala busuk akar cokelat pada tanaman jambu mete di Sumbawa, khususnya Kecamatan Pekat, Dompu-NTB diasosiasikan dengan serangan Phellinus noxius. Secara ilmiah jamur ini belum dapat dibuktikan patogensitasnya. Penelitian ini betujuan menguraikan hasil penelitian tentang uji patogenisitas isolat P. noxius pada bibit jambu mete dan 6 jenis tanaman berkayu lainnya. Penelitian dilakukan pada tahun 2003 di laboratoium dan rumah kaca Balai Penelitian Tanaman Rempah dan Obat. Isolat P. noxius diperoleh dari tanaman jambu mete sakit Kecamatan Pekat, Dompu-NTB, kemudian diperbanyak pada medium campuran beras jagung (1:1) dalam botol selai (vol. 250 ml.). Biakan inokulum jamur berumur salu bulan diinokulasikan pada pangkal batang dai tujuh jenis tanaman berkayu, yaitu: jambu mete (Anacardium occidentale) jenis Balakrisnan, kayu manis (Cinnamomum casia dan C. burmanii), kopi (Cofea arabtca), jarak pagar (Jatropa curcas). kapok (Ceiba pentandra), dan singkong (Manihot utilissima) yang ditumbuhkan di dalam kantong plastik. Hasil penelitian menunjukkan bahwa enam dai tanaman yang diinokulasi dengan P. noxius menghasilkan gejala penyakit daun menguning dan layu, sama sepeti gejala penyakit di lapangan. Bibit yang diinokulasi mati dalam waktu 2-3 minggu sampai dengan 2 bulan setelah inokulasi. Satu-satunya jenis tanaman yang menunjukkan gejala berbeda dan tidak mati, adalah singkong yang menunjukkan gejala kcrdil. Tanaman jambu mete dan jarak pagar merupakan tanaman inang yang baru untuk P. noxius. karena tanaman lainnya sudah pemah dilaporkan sebelumnya.. Mengingat ganasnya serangan P. noxius pada bibit yang diinokulasi maka kcwaspadaan perlu ditingkatkan untuk mencegah tersebamya penyakit ini ke daerah pengembangan mete lainnya di NTB.<br /><br />Kata kunci: Anacardium occidentale, jambu mete, Phellinus noxius. patogenisitas<br /><br /><strong>ABSTRACT</strong></p><p>Pathogenicity of Phellinus noxius isolatedfrom diseased cashew and other woody plants</p><p>Brown root rot symptom on cashew in Sumbawa, especially in Pekat Distict, Dompu - West Nusa Tenggara is associated with the attack of Phellinus noxius. The pathogenicity of this fungus has not been proven scientiically. This experiment was aimed to analyse the result of pathogenicity test of P. noxius isolate on the seedlings of cashew and 6 other woody plants. This research was done in 2003 in the laboratory and glass house of the Indonesian Spice and Medicinal Crop Research Institute. The P. noxius isolate was obtained from the infected cashew in Pekat District, Dompu - West Nusa Tcnggara, then multiplied in the mixture of rice and com medium in the 250 ml jam bottle. The one month fungus culture was inoculated on the stem base of the seven woody plants, namely cashew (Anacardium occidentale) Balakhrisnan cullivar, cinnamon (Cinnamommum casia and C. burmanii), coffee (Coffea arabica), castor (Jatropa curcas), kapok (Ceiba pentandra) and cassava (Manihot uilissima) grown in plastic pots. The result of this expeiment indicated that the six kinds of plants inoculated with /' noxius showed disease symptoms, such as wilting and yellowing of the leaves and died in about 2-3 weeks up to 2 months ater inoculation. The only plant that was not died but showed different symptom was cassava, its growth was very stunted but not died. The two plants, i.e. cashew and castor were new host plants for P. noxius. Considering the viciousness of /'. noxius atack on the inoculated seedlings, therefore the awareness to prevent the spread of this disease to other cashew plantations in West Nusa Tenggara should be raised.<br /><br />Key words : Anacardium occidentale. cashew, Phellinus noxius. pathogenicity</p>

Eksplorasi Rizobakteri Indigenous Dan Uji Antagonis Terhadap Patogen Rigidoporus microporus Dan Phellinus noxius Pada Tanaman Karet (Hevea brasiliensis) Secara In Vitro

Abstrak. Penelitian bertujuaan untuk mendapatkan isolat rizobakteri yang mampu berperan sebagai agens biokontrol terhadap pengendalian patogen R. microporus dan P. noxius secara in vitro serta sebagai agen rizobakteri pemacu pertumbuhan tanaman (RPPT). Penelitian dilaksanakan di Laboratorium Ilmu dan Teknologi Benih Jurusan Agroteknologi, Fakultas Pertanian, Universitas Syiah Kuala, Darussalam Banda Aceh, mulai Oktober sampai Desember 2017. Penelitian ini menggunakan Rancangan Acak Lengkap non faktorial. Faktor yang diteliti yaitu isolat rizobakteri, taraf yang dicobakan terdiri dari 15 isolat dan dua patogen antagonis R. microporus dan P. noxius, yang diulang sebanyak 3 kali sehingga terdapat 90 unit satuan percobaan. Hasil penelitian menunujukkan isolat rizobakteri mampu menekan pertumbuhan koloni cendawan patogen. Pada patogen uji R. microporus terdapat 3 rizobakteri yang paling baik yaitu isolat DLG5/3 dengan persentase penghambatan 68,33%, DLG4/1 dengan persentase penghambatan 66,66% dan DLG4/7 dengan persentase penghambatan 63,33%. Pada patogen uji P.noxius terdapat dua rizobakteri yang paling baik dalam menghambat pertumbuhan koloni cendawan patogen yaitu isolat DLG5/1 dengan persentase penghambatan 60,33%, dan DKP6/3 dengan persentase penghambatan 52,50%. Pada laju penghambatan isolat rizobakteri yang paling baik pada patogen R. microporus yaitu isolat DLG6/4 dan DKP4/1 dengan nilai rerata laju penghambatan 20,33 mm/hari. Pada patogen P. noxius menunjukan isolat rizobakteri yang paling baik yaitu isolat DLG4/1 dengan nilai laju penghambatan 12,05 mm/hari.Exploration of Indigenous Rizobacteria and Antagonistic Test against Patogen Rigidoporus microporus And Phellinus noxius In Rubber Plant (Hevea brasiliensis) In VitroAbstract. The research was conducted to obtain rhizobacteria isolates capable of acting as biocontrol agents on pathogen control of R. microporus and P. noxius in vitro and as plant growth promoter rhizobacteria (PGPR). The research was conducted at the Science and Technology of seed Laboratory, Department of Agrotechnology, Faculty of Agriculture, Syiah Kuala University, Darussalam Banda Aceh, starts from October to December 2017. The research using Completely Randomized Design non factorial. Factors researched were rhizobacteria isolates, the experimental stage consisted of 15 isolates and 2 antagonist were R. microporus dan P. noxius, and 3 time repeated until be found 90 units of treatment. The results showed that rhizobacteria isolates capable to inhibith growth of colonies pathogenic. In the pathogen of R. microporus test, there are 3 better rhizobacteria were DLG5/3 isolate with 68.33% inhibition percentage, DLG4/1 with 66.66% inhibition percentage and DLG4/7 with 63,33% inhibition percentage. In the pathogen of P. noxius there are 2 best rhizobacteria inhibiting growth, DLG5/1 isolate with 60.33% inhibition percentage, and DKP6/3 with 52,50% inhibition percentage. In the pathogen of R. microporus test, there are 2 better rhizobacteria were DLG6/4 and DKP4/1 with 20,33 mm/day inhibition percentage. In the pathogen of P. noxius test, the best rhizobacteria were DLG4/1 with 12,05 mm/day inhibition percentage.

Soil Is Not a Reservoir for Phellinus noxius

Phellinus noxius causes brown root rot (BRR) of diverse trees. Basidiospores and diseased host tissues have been recognized as important sources of P. noxius inoculum. This study aimed to understand whether P. noxius could occur or survive in soil without host tissues in the natural environment. Soil was sampled before and after the removal of diseased trees at eight BRR infection sites (total of 44 samples). No P. noxius colonies were recovered in soil plating assays, suggesting that no or little viable P. noxius resided in the soil. To know whether P. noxius could disseminate from decayed roots to the surrounding soil, rhizosphere and non-rhizosphere soils were sampled from another two infection sites. Although P. noxius DNA was detectable with specific primers, no P. noxius could be isolated, even from the rhizosphere soils around decayed roots covered with P. noxius mycelial mats. The association between viable P. noxius and the presence of its DNA was also investigated using field soil mixed with P. noxius arthrospores. After P. noxius was exterminated by flooding or fumigation treatment, its DNA remained detectable for a few weeks. The potential of onsite soil as an inoculum was tested using the highly susceptible loquat (Eriobotrya japonica). Loquats replanted in an infection site that had been cleaned up by simply removing the diseased stump and visible residual roots remained healthy for a year. Taken together, P. noxius is not a soilborne pathogen, and diseased host tissues should be the focus of field sanitation and detection for BRR.

In Vitro and in Planta Evaluation of Trichoderma asperellum TA as a Biocontrol Agent Against Phellinus noxius, the Cause of Brown Root Rot Disease of Trees

Brown root rot (BRR), caused by the white rot fungus Phellinus noxius, is an epidemic disease of diverse broadleaved and coniferous tree species in many tropical and subtropical regions. Flooding and trenching control measures are difficult to implement, and chemical controls can have an adverse impact on ecosystems. Previous studies have provided in vitro evidence for the potential use of Trichoderma spp. for biocontrol of BRR. Here, we analyzed the in vitro antagonistic and mycoparasitic abilities of four Trichoderma spp. isolates against four P. noxius isolates in dual culture and Ficus microcarpa wood blocks. A convenient inoculation system based on root inoculation of a highly susceptible loquat (Eriobotrya japonica) with P. noxius-colonized wheat-oat grains was developed to examine the effect of Trichoderma treatment in planta. Preventive application of Trichoderma asperellum TA, the isolate showing high antagonistic activity in vitro, was effective in preventing and delaying the wilting of P. noxius-inoculated loquat cuttings in greenhouse trials. To understand the specific niche in which T. asperellum TA interacts with P. noxius, KOH-aniline blue fluorescence microscopy was used to investigate the colonization of loquat roots by P. noxius and/or T. asperellum TA. Dilution plating assays were also conducted to quantify Trichoderma populations in the rhizosphere and potting mix. T. asperellum TA was able to robustly establish in the rhizosphere and potting mix but with scarce root penetration limited to the superficial layer. We discuss the timing and strategy for applying antagonistic Trichodema sp. on living trees or in BRR-infested areas for BRR management.

Evidence of Extensive Intraspecific Noncoding Reshuffling in a 169-kb Mitochondrial Genome of a Basidiomycetous Fungus

Comparative genomics of fungal mitochondrial genomes (mitogenomes) have revealed a remarkable pattern of rearrangement between and within major phyla owing to horizontal gene transfer and recombination. The role of recombination was exemplified at a finer evolutionary time scale in basidiomycetes group of fungi as they display a diversity of mitochondrial DNA inheritance patterns. Here, we assembled mitogenomes of six species from the Hymenochaetales order of basidiomycetes and examined 59 mitogenomes from 2 genetic lineages of Phellinus noxius. Gene order is largely collinear, while intergene regions are major determinants of mitogenome size variation. Substantial sequence divergence was found in shared introns consistent with high horizontal gene transfer frequency observed in yeasts, but we also identified a rare case where an intron was retained in five species since speciation. In contrast to the hyperdiversity observed in nuclear genomes of Phellinus noxius, mitogenomes’ intraspecific polymorphisms at protein-coding sequences are extremely low. Phylogeny network based on introns revealed turnover as well as exchange of introns between two lineages. Strikingly, some strains harbor a mosaic origin of introns from both lineages. Analysis of intergenic sequence indicated substantial differences between and within lineages, and an expansion may be ongoing as a result of exchange between distal intergenes. These findings suggest that the evolution in mitochondrial DNAs is usually lineage specific but chimeric mitotypes are frequently observed, thus capturing the possible evolutionary processes shaping mitogenomes in a basidiomycete. The large mitogenome sizes reported in various basidiomycetes appear to be a result of interspecific reshuffling of intergenes.