Fig 1.
A maximum likelihood phylogeny of 18S DNA sequences.
The phylogeny shows that Aphelenchoides besseyi are clustered together, and our sequences from different host plants can also be distinguished. The bootstrap values are indicated by numbers of percentage near each node, and a value higher than 90 are presented using red dots. GenBank ids of sequences include: EU196001.1 (Caenorhabditis elegans), AY508034.1 (Bursaphelenchus xylophilus isolate 186), KJ636306.1 (Bursaphelenchus xylophilus strain BursXyl1), AY284648.1 (Bursaphelenchus mucronatus), JQ348399.1 (Aphelenchus avenae), JQ957879 (Aphelenchoides blastophthorus), AY284643.1 (Aphelenchoides bicaudatus), JQ957890.1 (Aphelenchoides subtenuis), JQ957881.1 (Aphelenchoides ritzemabosi), AJ966475.1 (Aphelenchoides fragariae), and JQ957878.1 (Aphelenchoides besseyi isolate AChoBes1).
Fig 2.
The symptoms on the bird’s-nest fern leaves 21 days after inoculated with different host-origin Aphelenchoides besseyi isolates.
The bird’s-nest fern leaves inoculated with the (A) Fm, (B) Fsx and (C) Fgk isolates showed typical dark-brown patches. No symptoms were observed on bird’s-nest fern leaves inoculated with the rice-origin (D) Rl and (E) Rdg isolates. Leaves treated with Alternaria citri hyphal suspensions were used as (F) controls. The scale bars represent 10 cm.
Fig 3.
Amplified profiles of three bird’s-nest fern-origin isolates (Fm, Fsx and Fgk) and two rice-origin isolates (Rl and Rdg) of Aphelenchoides besseyi genomic DNA and cDNA with the GH45 and GH5 degenerate primer sets.
The GH 45 degenerate primers amplified two major bands of 548 and 439 bp (A) from Fm, Fsx and Fgk isolates and a 387 bp band from genomic DNA of the Rl and Rdg isolates. (B) A single band of 387 bp was amplified from cDNA of all test isolates. The GH5 degenerate primers amplified a major 491 bp band (C) from genomic DNA and (D) a 407 bp band from the cDNA of the Fm, Fsx and Fgk isolates.
Fig 4.
The gene structures of three GH45 genes in Aphelenchoides besseyi.
The introns not to exist in the Abe GH45-1 and presence in the Abe GH45-2 and Abe GH45-3 genes. Two introns, 163 bp and 142 bp, were found in the Abe GH45-2 and a 46-bp sized intron was found in the Abe GH45-3 gene.
Fig 5.
A maximum likelihood phylogeny of GH45 orthologues. The bootstrap support values are shown as percentage values near nodes, in which those higher than 90 are presented using red dots.
GenBank ids of sequences are described here: CCA72362.1 (Piriformospora indica), EHA54445.1 (Magnaporthe oryzae), ADV02790.1 (Rhizoctonia solani), ADZ99360.1 (Phialophora sp. CGMCC 3328), AEO65930.1 (Thielavia terrestris), ACV50414.1 (Cryptopygus antarcticus), AFD53063.1 (Bursaphelenchus mucronatus), ACD12136.1 (Bursaphelenchus xylophilus), and XP_008089512.1 (Colletotrichum graminicola M1.001).
Fig 6.
The protein and genomic DNA sequences of Abe GH5-1.
The Abe GH5-1 protein contains a SCP-like domain at the N-terminus and a GH5 cellulase domain at the C-terminus, as well as five introns were found in the Abe GH5-1 gene.
Fig 7.
A maximum likelihood phylogeny of nematode GH5 proteins. Orthologues of nematodes are represented using brown branches, and those of other species are shown as grey branches labeled with either green (bacteria) or blue (non-nematode eukaryotes) arcs.
Nematodes included in GH5_1 are Pristionchus pacificus (clade V). The phylogeny was computed using FastTree (as described in the Materials and Methods), and a high bootstrap support values of 0.91 reveals that GH5 orthologues of nematodes can be clearly distinguished from those of other eukaryotes and bacteria. For more details, please see S5 Fig.
Fig 8.
The amplification profiles of Aphelenchoides besseyi isolates; (A) Abe GH5-1 gene, (B) Abe GH45-2 and (C) Abe GH45-3.
(A) An Abe GH5-1 gene product of 1688 bp was amplified exclusively from the Fm, Fsx and Fgk isolates. Both Abe GH45-2 (B) and Abe GH45-3 (C) primers could amplify a major band from all five Aphelenchoides besseyi isolates.
Fig 9.
Identification of the Abe GH5-1 gene by Southern blotting.
Genomic DNA isolated from the five Aphelenchoides besseyi isolates were digested with Hinf I, and it was hybridized with an AbeFm-GH5-specific DNA probe.
Fig 10.
Localization of the Abe GH5-1 gene transcript in the Aphelenchoides besseyi by in situ hybridization with digoxigenin-labelled antisense (A and B) or sense probes (C).
The scale bars represent 20 μm. O: ovary, V: vulva, S: spicule and T: testis.