First report of Bursaphelenchus fungivorus (Nematoda: Aphelenchida) in Italy and an overview of nematodes associated with Crocus sativus L.

In September 2018, 20 corms of Crocus sativus were collected by the Plant Protection Service of Lazio Region on the farm “Arte Zafferano” located in Terracina (Latina, Lazio, Central Italy), in the karst area of Campo Soriano. The farm is characterized by labyrinths of calcareous rocks, dotted by small drained plots where saffron is cultivated. This survey was conducted to certify that these plants were free of plant-parasitic nematodes, such as Ditylenchus destructor (Thorne, 1945) and Ditylenchus dipsaci (Kühn, 1857; Filipjev, 1936), for their exportation of the corms to Guatemala (Central America).

Corms were put in polyethylene bags and then brought to the laboratory of Nematology at CREA-DC in Florence (Italy). These materials were kept in the refrigerator at about +4°C until they were processed for analysis. A mat of parallel fibers that shroud the corms (corm tunic) was removed and the naked corms were washed with sterilized water and then cut into 2 mm slices. Then the two materials were separately processed using the modified Baermann funnel method (Schindler, 1961). Isolated nematodes were observed with a dissection microscope to determine their presence and then under a light microscope for morphological identification at the genus or species level.

Specimens of Bursaphelenchus (Fuchs, 1937) were transferred to a 9 cm diameter Petri dish with 2% malt extract agar and Botrytis cinerea lawn in order to obtain a culture. After a two week incubation at 26°C, nematodes were collected for morphobiometrical and molecular studies.

Bursaphelenchus specimens were collected from the Petri dish, washed in sterilized water and heat-killed in warm water at 65°C; then they were fixed in triethanol-amine-formalin (Courtney et al., 1955), processed in glycerin by a modification of the glycerin-ethanol series of Seinhorst (1959) rapid method and finally, permanently mounted in anhydrous glycerin on glass microscope slides.

In total, 10 females and 10 males were photographed and measured. Photographs were taken with a Leica DM2000 light microscope using a Leica MC170 HD digital camera (Leica, Heerbrugg, Switzerland). Measurements were performed with the LEICA Application Suite (LAS) Version 4.9.0. Morphological and molecular characteristics were compared with the original description (Franklin and Hooper, 1962).

Two specimens of nematodes were individually put in 0.2 ml tube containing 50.0 µl InstaGene Matrix (BioRad), 1.5% SDS and 2.5 µl Proteinase K 20.0 µg/µl. The samples were incubated at 55.0°C for 3 hr, Proteinase K was inactivated by heating at 96.0°C for 10 min and DNA was recovered with alcoholic precipitation adding 100.0 µl of cold absolute ethanol. Pellets were air-dried and resuspended in 20.0 µl of double distilled water. The amplification of Internal Transcribed Spacer locus (ITS) was performed using conditions described in Burgermeister et al. (2005). The PCR products were sequenced at the Centro di Servizi per le Biotecnologie di Interesse Agrario Chimico e Industriale, University of Florence, Italy. Species identification was obtained through two phylogenetic trees (obtained with Neighbor Joining and Maximum likelihood algorithms) based on ITS locus and focused on fungivorous-group starting from sequences of this locus mined from GeneBank (Table 1). Alignments were computed with a local alignment algorithm (implemented in Kalign at EBI website) and the poorly aligned regions were removed with Gblocks v. 9.1b. The choice of appropriate substitution matrix for the data set was evaluated using Jmodeltest2 v. 2.1.10 considering AICc, BI criteria and DT method. Trees were computed using MEGA 7 software, choosing GTR +G +I as nucleotide substitution matrix and 1,000 bootstrap replicates.

No specimens belonging to the Ditylenchus genus were isolated from these samples, while some nematodes belonging to the Bursaphelenchus genus were extracted, but only from naked corms. Specimens presented the main morphological characteristics of Bursaphelenchus fungivorus: four lateral incisures, cephalic region offset by a constriction, stylet with weakly developed basal knobs; well-developed median bulb. Females present elongated conical tail with a rounded tip and ventrally bent. Vulva is without a flap in lateral and ventral views; both anterior and posterior vulval lips slightly protruding. In the males, tails are ventrally curved with compact spicules without cucullus (Fig. 1). The morphology of the Italian population of B. fungivorus (Table 2) agrees with the original description and with this species found in other countries (Braasch, 2001; Arias et al., 2005; Fonseca et al., 2014).

Figure 1:

The two nematodes vouchered had the same sequence in the ITS locus with a length of 969 bp, this sequence was submitted in GeneBank with accession number MK372853. Both trees obtained with maximum likelihood and neighbor joining methods share the same topology. The resulting tree (Fig. 2) showed a well-supported subdivision of fungivorus-group into two main clades, the first includes B. fungivorus, Bursaphelenchus seani (Giblin and Kaya, 1983), Bursaphelenchus arthuri (Burgermeister et al., 2005) and Bursaphelenchus arthuroides (Gu et al., 2012) while the second Bursaphelenchus thailandae (Braasch and Braasch-Bidasak, 2002), Bursaphelenchus parathailandae (Gu et al., 2012), Bursaphelenchus willibaldi (Schönfeld et al., 2006) and Bursaphelenchus braaschae (Gu and Wang, 2010). This finding is according to Kanzaki et al. (2016) where the same result was obtained starting from SSU and LSU combined sequences. Based on these results, we identified the nematodes as Bursaphelenchus fungivourss voucher GT2018.

Figure 2: