Development of a Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Phytopythium vexans

Brown root rot caused by Phytopythium vexans is a new destructive root disease on many plants such as Gingko, Citrus, kiwifruit, and ramie. The establishment of loop-mediated isothermal amplification (LAMP) technology for detecting P. vexans can help monitor and control brown root rot quickly, efficiently, and accurately. LAMP technology is known for its simplicity, sensitivity, and speed; and it does not require any specialized equipment – a water bath or a thermoblock is sufficient for isothermal amplifications. LAMP products can be visualized by using hydroxy naphthol blue (HNB) dye or agarose gel electrophoresis. In this study, by searching and comparing the internal transcribed spacer (ITS) sequences of P. vexans and the related species in oomycete genera Pythium, Phytopythium, and Phytophthora, we designed specific primers targeting the ITS gene region of P. vexans. Using HNB dye, we established a LAMP technique for rapid detection of P. vexans by visible color change. In addition, we optimized the protocol to enhance both sensitivity and specificity for P. vexans detection. Under the optimized condition, our protocol based on LAMP technology could detect as low as 24 copies of the P. vexans genomic DNA, which is ∼100 times more sensitive than conventional PCR. This method can successfully detect P. vexans using cell suspensions from P. vexans – infected ramie root tissues.

First Report of Alfalfa Root Rot Caused by Plectosphaerella cucumerina in Inner Mongolia Autonomous Region of China

Alfalfa (Medicago sativa L.) is an important forage crop with high nutrition for animal feed. In May 2016, a disease showing brown root rot was observed on alfalfa collected from several farms in Tongliao City (44°17′ N; 121°29′ E), Inner Mongolia Autonomous Region of China. The incidence of brown root rot was approximately 50 to 70% in the 2-year-old alfalfa field. Infected alfalfa exhibited varying degrees of decay in the tap root. Symptomatic roots were cut into 0.5-cm pieces, surface disinfected with 70% ethanol for 5 s and 0.1% HgCl2 for 35 s, then rinsed with sterilize distilled water three times, and placed onto potato dextrose agar (PDA) at 26°C in the dark. After 5 days, hyphal tips of the growing colonies were transferred onto PDA plates for purification. Forty-four isolates belonging to five fungal species were obtained from 20 diseased root samples. Six of the isolates resembled the genus Plectosphaerella. Colonies of these isolates were white to cream in color with sparse aerial mycelium, and then gradually became salmon pink with slimy or moist mycelium. The hyphae were transparent and branched. Colonies produced numerous hyphal coils with conidiophores. Conidiogenous cells and conidia were both hyaline, solitary, and smooth. Conidia were 4 to 8.5 ×1.2 to 4.8 µm (n= 100), 0 to 1 septum, elliptical and ovoid, and aggregating to form a head (Palm et al. 1995). According to these morphological characteristics, the fungus was identified as P. cucumerina (Lindf.) (Carlucci et al. 2012). To confirm the identification, the genomic DNA of two representative isolates was extracted and their internal transcribed spacer (ITS) region was amplified and sequenced with the primer pair ITS1/ITS4 (White et al. 1990). The ITS sequences of the two isolates were deposited in GenBank (acc. nos. MN915126 and MN915127). The two ITS sequences showed 99 to 100% identical to known P. cucumerina strains CBS 131739 (acc. no. KY662258.1) (Su et al. 2017) and MP313 (acc. no. KC756835.1) from alfalfa in China (Wen et al. 2015). To test for pathogenicity, a set of 15 alfalfa seedlings (cv. Aohan) were root-dipped in the conidial suspension of one of the isolates (1×105 conidia /ml) prepared from 7-day-old cultures on PDA. Inoculated seedlings were transplanted in three pots (10×15 cm) with sterilized nursery soil. Another set of five alfalfa seedlings inoculated with sterile water only served as the controls. Treated alfalfa seedlings were maintained in a greenhouse at 25°C to 28°C under a 12-h photoperiod. After 25 days, the roots of all inoculated plants showed brown lesions. P. cucumerina was reisolated from symptomatic tissue. No symptoms were observed on the control plants. P. cucumerina was previously reported on alfalfa in the fields of Huanxi Country (36°20′ N; 107°21′), Gansu Province, China (Wen et al. 2015). To our knowledge, this is the first report of P. cucumerina causing root rot of alfalfa in Inner Mongolia Autonomous Region, China. This disease may cause serious economic losses in the region. It is needed to develop effective management strategies for control of this disease.

Brown Root Rot Disease of Phyllanthus myrtifolius: The Causal Agent and Two Potential Biological Control Agents

Brown root rot (BRR), caused by Phellinus noxius (Corner) G. Cunningham, occurs on over 200 species of plants, especially woody trees and shrubs. Ceylon myrtle (Phyllanthus myrtifolius [Wight] Müll.Arg.), a common hedge plant, was recently observed to be infected with BRR. Disease diagnosis was performed by completing Koch’s postulates, and Ceylon myrtle was confirmed to be a new host of P. noxius. Typical symptoms of BRR were observed, including reduction in leaf size, dieback of branches, and suspended growth of young leaves. A disease severity index was used to quantify BRR in this study. Compared with Malabar chestnut, Ceylon myrtle was relatively resistant to BRR. Surprisingly, phylogenetic analysis of the ITS and 28S sequences revealed that isolates identified as P. noxius from Taiwan and many other countries were clustered in the same clade but separate from the clade comprising isolates from China, which were designated Pyrrhoderma noxium based on P. noxius. Therefore, to temporarily distinguish these pathogens, the former clade was designated GPN (global P. noxius), whereas the latter clade was designated CPN (China Py. noxium). In biocontrol assays, Streptomyces padanus and Bacillus sp. were selected for BRR control of Ceylon myrtle. Disease severity was reduced from 0.51 to 0.37 by S. padanus and to 0.14 by Bacillus sp. in greenhouse trials. In addition, the two biocontrol agents, especially S. padanus, exhibited good growth-promoting effects on cuttings of Ceylon myrtle. With these double advantages, S. padanus and Bacillus sp. have great potential to control BRR in practical applications.

Genome Sequence Resource for the Ramie Oomycete Pathogen Phytopythium vexans HF1

The oomycete Phytopythium vexans is a causative agent of patch canker, damping-off, and crown, stem, and root rot in many economically important plants. P. vexans HF1 was isolated in China, where it caused brown root rot of ramie, a fiber crop broadly cultivated in Asia. The genome of HF1 was sequenced by a combination of technologies producing short (Illumina HiSeq X) and long (PacBio RS) reads. The genome is 41.73 Mbp long, assembled into 44 contigs. It has a GC content of 58.17% and contains 13,051 predicted coding genes, including 1,461 putative virulence genes and 220 putative antimicrobial resistance genes. This genome sequence provides a resource for determining the molecular mechanisms of disease development in this pathosystem.

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>

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.