First Report of Fusarium concentricum as a Causal Agent of Fusarium Leaf Blotch on Pecan (Carya illinoinensis) in Southeast China

Abstract

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 β-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.