First report of Fusarium concentricum as a causal agent of Fusarium leaf blotch on pecan (Carya illinoinensis) in Southeast China.

The economically important nut crop pecan (Carya illinoinensis (Wangenh.) K. Koch) is seriously affected by increasing incidence of fungal disease worldwide (Xiao et al 2021). The top leaves of the pecan variety 'Pawnee' in the orchard of Zhejiang A&F University, Zhejiang, China were damaged by massive dark brown plaques in summer to autumn 2021. The causal agent was isolated from leaves with target plaques following the steps: sterilized with 70% alcohol (30 s × 2), rinsed with sterilized water (3 ×) before and after 5% sodium hypochlorite (30 s), excised the plaques, and placed on PDA medium at 28℃ in a dark incubator for 3-d. The mycelium on the edge of each colony was transferred to fresh SNA medium in dark for 2 weeks to induce conidia formation. A few conidia-germinated mycelia were transferredand inoculated on new plates containing fresh PDA medium to obtain the purified cultures. Koch's postulates were applied to validate the pathogenicity of the purified isolates. Non-woundedly healthy leaves (disinfected with 5% sodium hypochlorite) of 'Pawnee' seedlings were inoculated with 5 mm 7-d old purified cultures. Dark-brown spots appeared on the leaves 2 days post inoculation at 25℃. The spots became larger accompanied by partially cracking and slight deformation of inoculated leaves from day 2 to day 4, while the control leaves remained asymptomatic. A re-isolated strain ZJ-6 from these infected leaves was identified as the pathogenic isolate with the same symptom as the previous one. Morphologically, aerial mycelia of the pathogenic isolate ZJ-6 cashmere and white. The reverse of colony orange. The edge of the colony appeared gradually thinner, the aerial mycelia loose and flocky, and the matrix mycelium whitened. Hyphae were septate, translucent with smooth wall and 1.47-7.14 µm in width. Microconidia (n = 20) obovoid to fusoid, mainly with 0-septate, 4.45-7.78×4.79-16.25 µm. Macroconidia (n = 20) sickle, mainly with 3-5 septa, 5.56-10.28×56.67-114.54 µm. Simultaneous of monophialidic and polyphialidic conidiophores. Conidiophore width 1.47-3.68 µm, slightly smaller than vegetative hyphae. The morphological characteristics matched with previous descriptions of Fusarium species (Nirenberg and O'Donnell 1998; Wang et al 2013). The identity of ZJ-6 was confirmed by phylogenetic reconstruction using the concatenated sequences of the ATP citrate lyase (ACL1), Calmodulin (CaM), the internal transcribed spacer (ITS) rDNA region, ribosomal RNA gene (LSU), the largest subunit of DNA-dependent RNA polymerase II (RPB1), partial translation elongation factor-1 alpha (TEF) and ß-Tubulin (TUB). To this end, the genomic DNA of ZJ-6 was extracted by the M5 hipermix-MF859 (Mei5 Biotechnology) and submitted to GenBank under the accession numbers OP933646, OP933647, OP925890, OP925889, OP933396, OP933648, and OP933397, respectively. The obtained sequences of ZJ-6 were used for nucleotide BLAST against thetandard databases, respectively, and the strains with sequence identification values above 98% were selected to construct multiple alignment for building a phylogenetic tree. This analyses allowed the identification of ZJ-6 as Fusarium concentricum Nirenberg & O'Donnell, a species with few reports that can cause serious damage to the fruits and branches of other hosts (Hasan et al 2020; Huda-Shakirah et al 2020; Wang et al 2013). This is the first report of pathogenic F. concentricum on pecan in Southeast China that caused no harvest of infected plants.

First report of Colletotrichum plurivorum causing anthracnose on pecan (Carya illinoinensis) in China.

Pecan (Carya illinoinensis) is an economically important nut crop worldwide (Xiao et al 2021). Anthracnose symptoms were found on pecan fruits and leaves in plantations in Anhui and Jiangsu provinces, China in August 2019. Irregular, dark brown or black spotted lesions firstly appeared on the surface and inside of fruits, and spread to all leaves. The symptoms resulted in 30% to 50% leaf drop and nearly a half of fruit decay in almost all trees of the susceptible cv. Wichita. The causal agent were isolated from fruits with target symptoms following the steps: surface disinfected with 75% ethanol (2×, 30 s), rinsed with sterile deionized water (3×), ~ 0.5 cm small fragments of the fruits excised and plated on potato dextrose agar (PDA) medium and incubated at 28 °C in dark for 3-d. Mycelium of each colony was picked and incubated on fresh PDA at 25 °C with a 12-hour light/dark cycle for 6-d to induce conidia formation. One 5-mm hyphal plug produced from each single spore isolate was transferred onto fresh PDA to obtain the pure cultures. Koch's postulates was employed for pathogenicity determination of the isolates. Non-wounded healthy leaves from seedlings of the disease susceptible cv. Pawnee were disinfected with 1% NaClO and inoculated with 5-mm 5-d hyphal of each isolate at 25 ℃. Tiny lesion spots were visible on the leaves after 2 days post inoculation (DPI) with isolate W-6 (the only pathogenic one among all isolates), and expanded over time until to the leaves withered, while the control leaves and leaves inoculated with other isolates remained asymptomatic. The pathogenicity of W-6 were confirmed using leaves and fruits of living Pawnee trees growing in Linglong Mountain Plantation, Lin'an, Hangzhou, China (119°38'51″E, 30°12'39″N, elevation: 119m). Three experimental replicates were conducted separately with three bio-replicates for all pathogenetic testing. The same symptoms were observed on both detached leaves and leaves and fruits of living trees.. The colony of W-6 have round cottony mycelium with complete edges and showed the fastest growth rate 3 - 4 DPI. After 7 DPI, white aerial mycelium turned yellowish brown and formed Acervulus in the mycelium. Conidia (n=50) one-celled, 12.0 - 20.0 µm × 3.5 to 6.0 µm width. Hyaline cylindrical with slightly rounded ends and two or three large guttulate at the centre. Most Acervulus dark brown and slightly irregular in shape, 12.70 × 18.79 µm (n=10). Setae were dark brown in color with average length around 34.10 µm (n=10). These characteristics matched previous descriptions of Colletotrichum orchidearum species complex, including C. plurivorum (Damm et al 2019). The identity of W-6 was confirmed by multi-locus phylogenetic analysis using the internal transcribed spacer (ITS) rDNA region and partial sequences of the conserved genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), beta-tubulin 2 (TUB2), and chitin synthase (CHS). The sequences of W-6 were used for Basic Local Alignment Search Tool (BLAST) against NCBI GenBank and the sequences with 100% identity to that of W-6 were achieved, respectively. The concatenated sequences of the ACT-CHS-GAPDH-ITS-TUB2 was used for building a phylogenetic tree. The molecular analyses allowed the identification of the pathogen as C. plurivorum. It was known that 9 of the 11 Colletotrichum species causing pecan anthrax worldwide were reported in southern China (Brenneman 1989; Oh et al 2021). This is the first report of C. plurivorum as causal agent of pecan in China.

Comparative plastomes of Carya species provide new insights into the plastomes evolution and maternal phylogeny of the genus.

Carya, in the Juglandiodeae subfamily, is to a typical temperate-subtropical forest-tree genus for studying the phylogenetic evolution and intercontinental disjunction between eastern Asia (EA) and North America (NA). Species of the genus have high economic values worldwide for their high-quality wood and the rich healthy factors of their nuts. Although previous efforts based on multiple molecular markers or genome-wide SNPs supported the monophyly of Carya and its two EA and NA major subclades, the maternal phylogeny of Carya still need to be comprehensively evaluated. The variation of Carya plastome has never been thoroughly characterized. Here, we novelly present 19 newly generated plastomes of congeneric Carya species, including the recently rediscovered critically endangered C. poilanei. The overall assessment of plastomes revealed highly conservative in the general structures. Our results indicated that remarkable differences in several plastome features are highly consistent with the EA-NA disjunction and showed the relatively diverse matrilineal sources among EA Carya compared to NA Carya. The maternal phylogenies were conducted with different plastome regions and full-length plastome datasets from 30 plastomes, representing 26 species in six genera of Juglandoideae and Myrica rubra (as root). Six out of seven phylogenetic topologies strongly supported the previously reported relationships among genera of Juglandoideae and the two subclades of EA and NA Carya, but displayed significant incongruencies between species within the EA and NA subclades. The phylogenetic tree generated from full-length plastomes demonstrated the optimal topology and revealed significant geographical maternal relationships among Carya species, especially for EA Carya within overlapping distribution areas. The full-length plastome-based phylogenetic topology also strongly supported the taxonomic status of five controversial species as separate species of Carya. Historical and recent introgressive hybridization and plastid captures might contribute to plastome geographic patterns and inconsistencies between topologies built from different datasets, while incomplete lineage sorting could account for the discordance between maternal topology and the previous nuclear genome data-based phylogeny. Our findings highlight full-length plastomes as an ideal tool for exploring maternal relationships among the subclades of Carya, and potentially in other outcrossing perennial woody plants, for resolving plastome phylogenetic relationships.

First report of Curvularia muehlenbeckiae, the causal agent of Curvularia leaf spot on pecan (Carya illinoinensis) in China.

The pecan (Carya illinoinensis) industry is largely affected by the increased incidence of diseases (Xiao et al 2021). Leaf spot symptoms were identified in an orchard of cultivar Pawnee pecan trees at Zhejiang A&F University, Zhejiang, China in August 2020. Small black spots occurred on the veins and edges of the leaves and nearby tissues turned yellow and slightly deformed (May to July). The spots (0.5-1.5cm) spread to all leaves with 25% to 40% leaf drop occurring in almost all trees between August and October. The causal pathogen was isolated from leaves with target symptoms using the following method: surface sterilized with 70% alcohol (2×, 30 s), rinsed with sterilized water (3×), leaf spots excised and placed on PCA media, and left to incubate at 28℃ in the dark for 3-d. Mycelium on the edge of each clone was excised and incubated on fresh oatmeal agar medium with a 12-hour light/dark cycle for 7-d to obtain conidia. Single spore isolates were germinated on PDA medium under the same conditions as previously described, one 5-mm hyphal plug was transferred to fresh PCA media to obtain the pure cultures. The pathogenicity of the isolates were verified using Koch's postulates. Non-wounded healthy leaves (disinfected with 1% NaClO) of cv. Pawnee (disease susceptible) were obtained from seedlings grown in green-house at 26 ℃ and inoculated with 5-mm hyphal plugs and a conidia-hyphae suspension (~106/mL) containing one-week old purified cultures. After 3-15 days post-inoculation, small black spots appeared on the leaves inoculated with isolate P-6 (the only pathogenic isolate from the leaf spots in the orchard) and grew larger until the whole leaf wilted while the control leaves remained asymptomatic. The experiment was repeated two times with two bio-replicates each run. Finally, the pathogen was re-isolated from infected leaves, which showed the same symptoms as the previous isolate. Aerial mycelia of P-6 turned from white to gray and substrate mycelia from brown to black. Colonies had a fimbriate margin before mycelia filled the medium. Hyphae were septate, branched, brown or black, smooth wall and 1.4-10 µm in width. Conidiophore single-branch, dark brown, curved or straight, 1.93-5.51×44.12-104.41 µm width, conidiogenous cells 6.66-16.67 µm (terminal) and 8.82-23.33 µm (intercalary) length, mono- to polytretic, proliferating sympodially. Conidia (n=20) four cells, 15.7 - 25.7 µm × 7.1 - 11.4 µm wdth, swelling and curving from the basal cell to the third. The bending angle was 5° to 80°. The middle two cells were brown and usually verruculose, the basal and apical cells paler and less ornamented. No sexual morph observed. The morphological characteristics matched previous descriptions of Curvularia species (Madrid et al 2014). The identity of P-6 was confirmed by phylogenetic reconstruction using the concatenated sequences of ITS rDNA, partial GAPDH, LSU, TEF-1α and RPB2 regions (Raza et al 2019). The genomic DNA of P-6 was extracted by the M5 hipermix-MF859 (Mei5 Biotechnology). The sequences of P-6 were used for nucleotide BLAST against the Standard databases and model strains were selected to construct the concatenated sequences of GAPDH-ITS-LSU-TEF-1α-RPB2 for building a phylogenetic tree. This analysis identified P-6 as a strain of C. muehlenbeckiae, a species with few reports other than in gramineous crops (Raza et al 2019; Chen et al 2021; Cui et al 2020; Ni et al 2016). This is the first report of C. muehlenbeckiae on pecan in China and worldwide.

Chromosome-scale assembly reveals asymmetric paleo-subgenome evolution and targets for the acceleration of fungal resistance breeding in the nut crop, pecan.

Pecan (Carya illinoinensis) is a tree nut crop of worldwide economic importance that is rich in health-promoting factors. However, pecan production and nut quality are greatly challenged by environmental stresses such as the outbreak of severe fungal diseases. Here, we report a high-quality, chromosome-scale genome assembly of the controlled-cross pecan cultivar 'Pawnee' constructed by integrating Nanopore sequencing and Hi-C technologies. Phylogenetic and evolutionary analyses reveal two whole-genome duplication (WGD) events and two paleo-subgenomes in pecan and walnut. Time estimates suggest that the recent WGD event and considerable genome rearrangements in pecan and walnut account for expansions in genome size and chromosome number after the divergence from bayberry. The two paleo-subgenomes differ in size and protein-coding gene sets. They exhibit uneven ancient gene loss, asymmetrical distribution of transposable elements (especially LTR/Copia and LTR/Gypsy), and expansions in transcription factor families (such as the extreme pecan-specific expansion in the far-red impaired response 1 family), which are likely to reflect the long evolutionary history of species in the Juglandaceae. A whole-genome scan of resequencing data from 86 pecan scab-associated core accessions identified 47 chromosome regions containing 185 putative candidate genes. Significant changes were detected in the expression of candidate genes associated with the chitin response pathway under chitin treatment in the scab-resistant and scab-susceptible cultivars 'Excell' and 'Pawnee'. These findings enable us to identify key genes that may be important susceptibility factors for fungal diseases in pecan. The high-quality sequences are valuable resources for pecan breeders and will provide a foundation for the production and quality improvement of tree nut crops.