Sacha inchi (Plukenetia volubilis L.) belongs to the family Euphorbiaceae. It is a perennial wooden oilseed crop, and also exhibits a good source of polyunsaturated fatty acids, protein and other bioactive compounds, such as tocopherols, carotenes and phytosterols (Chirinos et al. 2013). During 2017-2018 survey, canker disease showing greyish-brown sunken lesions was observed on the branches of sacha inchi in Danzhou campus, Hainan University, China. The disease incidence is less than 5%. However, it can lead to leaf yellowing, wilt, and eventually the whole plant death. In Nov. 2017, twelve branches showing the typical canker symptoms were collected and covered with parafilm at both ends of all samples to prevent desiccation and placed in black plastic bags keeping at 4°C until isolations were made. Samples were rinsed with tap water and dried with paper towels. Fragments, 5mm in length and cut from the junction of diseased and healthy parts of branches, were surface-sterilized with 2% sodium hypochlorite solution for 2 min, rinsed with sterilized distilled water for 5 times, dried by sterilized filter paper, plated on PDA medium amended with 100 μg/mL streptomycin (PDA-str) and incubated in the dark for 4 days at 28°C. Pure cultures of fungal isolates were obtained by transferring mycelial fragments from colony margins onto fresh PDA plates and incubated as described before. The colonies of cultures were initially white, and eventually turned black after 4 days on PDA medium (Fig S1A). The morphology characterization of conidia produced by the isolates was initially hyaline and aseptate (Fig S1B), and a single median septum formed in the mature conidia (Fig S1B). The average size of 50 conidia was 16.39±1.46ⅹ 8.52±0.92μm for J6, and 15.64±1.73ⅹ 8.94±0.86μm for J3. Three genes were used for phylogenetic analysis (Alves et al. 2006). ITS regions and the partial of TUB (β-tubulin gene) were amplified using the primer pairs ITS1 and ITS4 (White et al. 1990), Bt2a and Bt2b (Glass and Donaldson 1995), respectively, and EF1-688F/EF1-1251R for J3 and EF1-728F/EF1-986R for J6 were used to amplify TEF (translation elongation factor 1-alpha) (Alves et al. 2008). The sequences of ITS, TUB and TEF from J3 and J6 were deposited in Gene-Bank (Table S1). The blast searches in Gene-Bank with ITS, TUB and TEF amplified from isolates J3, respectively, revealed 100, 99, and 100% identities with L. pseudotheobromae, and isolate J6 showed 100, 100 and 99% of identity with L. theobromae. The phylogenetic analysis of the combined ITS, TUB and TEF sequences of J3, J6 and 28 reference strains retrieved from Gene-Bank was performed using the program MEGA 6.0 evaluated by 1000 bootstrap replications, and the result was consistent with the conclusion above (Fig S2). With the phylogenic studies supported by morphological characters, J3 was identified as L. pseudotheobromae and J6 was L. theobromae. For the pathogenicity test, J3 and J6 were used to inoculate 4-week-old healthy sacha inchi potted seedlings. One wound about 5 mm in depth per seedling stem was made using a sterile blade. A 5-mm-diameter mycelium plug of each isolate taken from the edge of 4-day-old culture growing on PDA was placed to the freshly wound of each plant stem and the inoculated area was wrapped with Parafilm. Sterile PDA plugs were placed onto the wounds of control seedlings. Nine healthy seedlings were inoculated with each isolate or PDA plugs in a completely randomized design. After inoculation, plants were placed in a greenhouse at room temperature (26 to 30°C, 80% RH) and were irrigated when needed. The experiment was conducted twice. Five days later, black or dark-brown canker lesions formed on the stems of inoculated plants, and expended upward and downward from the inoculation points. Pycnidia produced on the necrotic regions and were used to to observe the morphology of conidia (Fig S3). The fungus L. pseudotheobromae or L. theobromae can be re-isolated from the inoculated plants, but not from the control ones. L. pseudotheobromae was recorded to be collected from dead leaves of P. volubilis in Yunnan Province, China, but did not prove this fungus to be pathogenic (Tennakoon et al. 2016). This is the first report that L. theobromae and L. pseudotheobromae causing stem canker in sacha inchi in Hainan, China. The results pave the way for the development of management strategies for canker disease in sacha inchi.

MicroRNA-like small RNAs (milRNAs) and their regulatory roles in the interaction between plant and fungus have recently aroused keen interest of plant pathologists. Trichoderma spp., one of the widespread biocontrol fungi, can promote plant growth and induce plant disease resistance. To investigate milRNAs potentially involved in the interaction between Trichoderma and tomato roots, a small RNA (sRNA) library expressed during the interaction of T. asperellum DQ-1 and tomato roots was constructed and sequenced using the Illumina HiSeqTM 2500 sequencing platform. From 13,464,142 sRNA reads, we identified 21 milRNA candidates that were similar to other known microRNAs in the miRBase database and 22 novel milRNA candidates that possessed a stable microRNA precursor hairpin structure. Among them, three milRNA candidates showed different expression level in the interaction according to the result of stem-loop RT-PCR indicating that these milRNAs may play a distinct regulatory role in the interaction between Trichoderma and tomato roots. The potential transboundary milRNAs from T. asperellum and their target genes in tomato were predicted by bioinformatics analysis. The results revealed that several interesting proteins involved in plant growth and development, disease resistance, seed maturation, and osmotic stress signal transduction might be regulated by the transboundary milRNAs. To our knowledge, this is the first report of milRNAs taking part in the process of interaction of T. asperellum and tomato roots and associated with plant promotion and disease resistance. The results might be useful to unravel the mechanism of interaction between Trichoderma and tomato.

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Curvularia lunata (Wakker) Boed, the causal agent of leaf spot in maize, is prone to mutation, making it difficult to control. RNAi technology has proven to be an important tool of genetic engineering and functional genomics aimed for crop improvement. MicroRNAs (miRNAs), which act as post-transcriptional regulators, often cause translational repression and gene silencing. In this article, four small RNA (sRNA) libraries were generated from two maize genotypes inoculated by C. lunata; among these, ltR1 and ltR2 were from the susceptible variety Huangzao 4 (HZ), ltR3 and ltR4, from the resistant variety Luyuan (LY), and 2286, 2145, 1556 and 2504 reads were annotated as miRNA in these four sRNA libraries, respectively. Through the combined analysis of high-throughput sequencing, microarray hybridization and degradome, 48 miRNAs were identified as being related to maize resistance to C. lunata. Among these, PC-732 and PC-169, two new maize miRNAs discovered, were predicted to cleave mRNAs of metacaspase 1 (AMC1) and thioredoxin family protein (Trx), respectively, possibly playing crucial roles in the resistance of maize to C. lunata. To further confirm the role of PC-732 in the interaction of maize and C. lunata, the miRNA was silenced through STTM (short tandem target mimic) technology, and we found that knocking down PC-732 decreased the susceptibility of maize to C. lunata. Precisely speaking, the target gene of PC-732 might inhibit the expression of disease resistance-related genes during the interaction between maize and C. lunata. Overall, the findings of this study indicated the existence of miRNAs involved in the resistance of maize to C. lunata and will contribute to rapidly clarify the resistant mechanism of maize to C. lunata.