First report of brown leaf spot on Juglans sigillata caused by Ophiognomonia leptostyla in Sichuan, China

Juglans sigillata Dode (Iron walnut) is mostly distributed in southwestern China, and valued for wood and nuts (Feng et al. 2018). In April 2020, we surveyed a walnut garden located in Baisha Town, Wanyuan City, (Sichuan, China), where brown spot symptoms were observed on leaves of ten trees among of 100 plants, and this disease can result in a reduced growth potential when trees are severely infected. Necrotic and subcircular lesions with conidiamata were observed on diseased leaves. Symptomatic leaves were collected and taken back to the laboratory forfurther analysis. Using the single spore isolation technique developed by Chomnunti et al. (2014), five isolates were grown from the infected leaves on Potato Dextrose Agar medium (PDA). The five isolates had similar colony morphology, which was initially white, suborbicular, gradually turning yellowish with black spots, developing fluffy aerial mycelium. Morphological characteristics were examined using light microscopy on the PDA. Conidiogenous cells were subcylindrical to cylindrical, or ampulliform, hyaline, rarely branched. Macroconidia were lunate, reniform, hyaline, 1-3-septate, mostly 1-septate, distinctly constricted at the septum, the basal cell was bluntly rounded, the apical cell had an acute end, and the basal cell was equal to or larger than the apical cell, measuring 22 to 40.5 × 2.5 to 8.3 μm (mean = 32 × 6.2 μm, n = 50). Microconidia were botuliform, or subfusiform, hyaline, both ends were rounded, straight or curved, aseptate, and measured 10 to 28.5 × 1.9 to 3.7 μm (mean= 17.2 × 2.7 μm, n = 20). A multilocus approach was conducted for precise identification of a representative isolate SICAUCC 20-0012. The internal transcribed spacer regions (ITS), guanine nucleotide-binding protein subunit beta gene (MS204), and translation elongation factor 1-alpha (tef1-α) of isolate SICAUCC 20-0012 were amplified and sequenced as described by Sogonov et al. (2008) and Walker et al. (2012a). GenBank Accession Nos. for ITS, MS204, and tef1-α are MW250303, MW246773, and MW246775, respectively. Phylogenetic analyses showed 100% support with Ophiognomonia leptostyla (Fr.) Sogonov, and the morphology was consistent with the asexual stage of O. leptostyla documented by Walker et al. (2012b). To test Koch’s postulates, five healthy plants of J. sigillata (2- to 3-year-old) with 5-8 leaves per plant were inoculated with conidial suspensions (104 conidia/mL) after wounded with a small pin as described by Desai et al. (2019), and the same number of healthy plants were wounded and sprayed with sterile distilled water as controls. Plants were sprayed regularly with distilled water every day and placed in a growth chamber at 25℃ with a 12-h fluorescent light/dark regimen. After 15 days, typical brown spot symptoms developed on inoculated leaves, but not on the controls. The fungus O. leptostyla was reisolated from the lesion as described above but not from non-inoculated leaves. O. leptostyla has been reported on some walnut trees; for example: J. ailantifolia, J. californica, J. cinerea, J. hindsii, J. major, J. mandshurica, J. nigra, and J. regia (Farr & Rossman 2020). However, to our knowledge, this is the first report of O. leptostyla causing brown leaf spot on J. sigillata. J. sigillata is an economically important tree in southwest China, and fungicide treatments should be considered to prevent the spread of this fungus before it becomes more widespread. Chunlin Yang, Yu Deng, and Feihu Wang contributed equally to this work. This research was supported by the Key Research and Development Project of Sichuan Province (2021YFYZ0032)

Stem Rot Disease of Juglans sigillata Dode Caused by Fusarium fujikuroi in China

Juglans sigillata Dode is an endemic species in the southwest China, and is an important nut and woody oil tree. The shell of its fruit is hard and can be used to make various crafts. From 216 to 2019, typical stem rot symptoms of 8-year-old J. sigillata were observed in cultivated fields in a 600-ha orchard in Zigong, Sichuan province, China. At this orchard, approximately 35% of the trees have been seriously damaged over the past few years. The typical symptoms were water-soaking on the stem, rotting, wilting, and blighting, eventually leading to the death of the plant. In June, ten diseased tissues were collected and surface-sterilized by 3% NaClO and 75% alcohol. Morphological observations were made from the isolates grown on Potato dextrose agar (PDA) and incubated at 25°C for 3 to 9 days. Morphological characteristics were made on pure cultures grown on Synthetic low nutrient agar (SNA). Five isolates with similar morphology were isolated from single spores. Colonies on PDA reached 8.3 cm in diameter after 6 days at 25 °C, aerial mycelia were white to cream and wol-like, later turning violet and dark purple with age. The hyphae of the strain were colorless and septate. There were two types of conidia on SNA, microconidia and macroconidia. Microconidia (n = 50) were oval, elliptic or clavate, no septate, 2.2 to 3.8 × 7.6 to11.7 μm. Conidiophores were branched or unbranched, solitary or in groups, phialides cylindrical to flask-shaped, monophialidic and polyphialidic. Macroconidia (n = 50) were long slender with a curved apical cell and foot-like basal cell, 3 to 4 septate and 2.1 to 3.9 × 26.2 to 53.4 μm. For molecular identification, the internal transcribed spacer (ITS), β-tubulin (TUB2), translation elongation factor (TEF1) and large subunit (LSU) were amplified with the corresponding primer pairs ITS1/ITS4 (White et al. 1990), BT2A/BT2B, EF1/EF2 (O’Donnell et al. 1997), and LROR/LR5 (Rehner and Samuels 1994), respectively. BLAST search results indicated that the ITS, TUB2, TEF1, LSU sequences (GenBank acc. nos. MT791384, MT786729, MN853324, and MT705246) showed 99 to100% identity with Fusarium fujikuroi sequences at NCBI (GenBank acc. nos. MG798789, MH398245, MK604519 and KJ954504). The results were confirmed by multilocus phylogenetic analysis. Based on the morphological characteristics and molecular analysis of the isolates, the fungus was identified as F. fujikuroi (Leslie and Summerell 2006). Koch’s postulates were checked under controlled conditions. Fifteen 2-year-old healthy potted J. sigillata were inoculated by pricking the epidermis of stem with a needle and applying 150 µl of a microconidial suspension (1 × 106 spores/ml) to the wounded surface with a brush. Sterilizd distilled water was used as the control. The experiment was repeated three times. All the plants were incubated at 25 ± 2°C after inoculation for daily observation of disease development. After 12 days, the inoculated plants showed the same symptoms as observed in the original diseased plants, while the control plants were asymptomatic. The fungus was re-isolated from the symptomatic stems and was completely identical to the isolates used to inoculate the plants. Thus, we confirmed that F. fujikuroi caused the stem rot of J. sigillata. To our knowledge, this is the first report of this fungus causing stem rot in J. sigillata in China. Our results can help identify stem rot disease of J. sigillata and develop control measures for the disease.

Genetic diversity of Juglans sigillata Dode germplasm in Yunnan Province, China, as revealed by SSRs

Iron walnut, Juglans sigillata Dode, restricted to southwestern China, has its centre of distribution in Yunnan Province which has a varied climate, geography and rich plant diversity. Yunnan contains abundant J. sigillata germplasm. In this study, a provincial-scale set of walnut germplasm resources (14 populations comprising 1122 individuals) was evaluated for genetic diversity based on 20 simple sequence repeat (SSR) loci. The number of SSR alleles per locus ranged from 7 to 27, with an average of 17.55. Mean allelic richness and mean private allelic richness ranged from 3.40 to 4.62 and 0.11 to 0.36, with average of 3.93 and 0.30, respectively. Expected heterozygosity (He) varied from 0.26 to 0.78, with an average of 0.57. Polymorphism information content ranged from 0.22 to 0.79, with an average of 0.57. Genetic differentiation (FST) was 0.05, indicating that only 5% of total genetic variability was inter-populational, a finding supported by an analysis of molecular variance and STRUCTURE analysis. Relatively high gene flow (Nm = 6.70) was observed between populations. A unweighted pair-group method with arithmetic analysis classified the 14 populations into two major groups. Mantel testing uncovered a significant correlation between geographic distance and genetic distance (r = 0.33, P = 0.04). Overall, the research revealed a moderately high level of genetic diversity in the germplasm and low genetic differentiation among populations, which showed great potential for further development and exploitation of this resource.

Chromosomal-level assembly of Juglans sigillata genome using Nanopore, BioNano, and Hi-C analysis

Background Juglans sigillata, or iron walnut, belonging to the order Juglandales, is an economically important tree species in Asia, especially in the Yunnan province of China. However, little research has been conducted on J. sigillata at the molecular level, which hinders understanding of its evolution, speciation, and synthesis of secondary metabolites, as well as its wide adaptability to its plateau environment. To address these issues, a high-quality reference genome of J. sigillata would be useful. Findings To construct a high-quality reference genome for J. sigillata, we first generated 38.0 Gb short reads and 66.31 Gb long reads using Illumina and Nanopore sequencing platforms, respectively. The sequencing data were assembled into a 536.50-Mb genome assembly with a contig N50 length of 4.31 Mb. Additionally, we applied BioNano technology to identify contacts among contigs, which were then used to assemble contigs into scaffolds, resulting in a genome assembly with scaffold N50 length of 16.43 Mb and contig N50 length of 4.34 Mb. To obtain a chromosome-level genome assembly, we constructed 1 Hi-C library and sequenced 79.97 Gb raw reads using the Illumina HiSeq platform. We anchored ∼93% of the scaffold sequences into 16 chromosomes and evaluated the quality of our assembly using the high contact frequency heat map. Repetitive elements account for 50.06% of the genome, and 30,387 protein-coding genes were predicted from the genome, of which 99.8% have been functionally annotated. The genome-wide phylogenetic tree indicated an estimated divergence time between J. sigillata and Juglans regia of 49 million years ago on the basis of single-copy orthologous genes. Conclusions We provide the first chromosome-level genome for J. sigillata. It will lay a valuable foundation for future research on the genetic improvement of J. sigillata.