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Introduction
Xylella fastidiosa is a phytopathogenic bacterium that causes important diseases in different crops such as Pierce´s disease of grapevine (Vitis vinifera), coffee leaf scorch in coffee (Coffea arabiga), quick decline syndrome of olive (Olea europea), almond leaf scorch (Prunus dulcis), the citrus variegated chlorosis, alfalfa dwarfism (Medicago sativa) and leaf scorch in urban trees (de Lima et al., 1998; Purcell et al., 1999; Li, et al., 2001; Almeida & Purcell, 2003; Sisterson et al., 2010; Costa et al., 2004). The bacterium has been found dormant in asymptomatic hosts such as california mugwort (Artemisia douglasiana) and barnyard grass (Echinochloa cruz-galli) that serve as inoculum reservoirs (Hopkins & Purcell, 2002). The symptoms presented by infected plants with X. fastidiosa usually appear first in some leaves and then in almost all the foliage. The affected area is delineated by a narrow chlorotic halo that becomes especially clear in autumn. It presents premature defoliation and malformation in new leaves, the fruits grow abnormally, the stems can show internal and external discoloration; in some cases, there is a regressive death and abnormal growth eventually ending in host death (Janse & Obradovic, 2010). X. fastidiosa spreads naturally by insect vectors mainly leafhoppers (Cicadellidae) and spittlebugs (Cercopidae) very fast because there is no latency period for transmission, some related species as X. fastidiosa vectors are: red head leafhopper (Xyphon fulgidum) (Catanach et al., 2013), green leafhopper (Draeculacephala minerva), blue-green leafhopper (Graphocephala atropunctata), alfalfa leafhopper (Phera lacerta) (Burks & Redak, 2003a), glassy winged leafhopper (Homalodisca vitripennis) and Oncometopia nigricans, among others (Hill and Purcell, 1997; Brlansky et al., 2002; Díaz, 2003; Hoddle, 2004; Blackmer, 2006; Daane et al., 2011). The bacterium is not trans-ovarial or trans-stag, it feeds and reproduces in salivary of adult insects which can remain infective throughout their lives (Almeida et al., 2005; Redak et al., 2004). The bacterium detection is mainly carried out by serological techniques such as ELISA and molecular techniques such as PCR in which specific primers are used, such as RST3/; RST33 (Minsavage et al., 1994) and XF1968-R/XF1968-L (Firrao & Bazzi, 1994). The objective of present investigation was to determine the vectors and hosts of Xylella fastidiosa in wine-growing zone of Parras, Coahuila, Mexico.
Material and Methods
Plant material collection
The plant material collection was carried out in area surrounding 4 vineyards in Parras, Coahuila, Mexico during 2016. Leaves and shoots of 22 plant species with symptoms like to those caused by X. fastidiosa were collected.
Insect collection
By using an entomological striking net through a series of 500 nets carried out in periphery of vineyards, insects were collected on the weeds and were also manually collected in bushes, fruit tres and forest. The insects were preserved in ethanol 70 %.
Insects identification
The insects were separated and grouped by morphological observations, the identification was made with help of taxonomic keys (DeLong & Davidson, 1935; Young, 1958; Blocker, 1967) and male genitalia extraction.
DNA extraction from plants and insects
For this investigation, the CTAB DNA extraction method (Doyle & Doyle, 1987) with modifications was used. 50 to 100 mg of tissue were taken from each plant and in the case of insects, the whole specimen was considered. From plants, the main veins and the petiole of each leaf were selected, these were macerated with 300 μL of CTAB buffer, placed in eppendorf tubes and 1 mL of CTAB buffer was added, 100 μL of 5M NaCl was added, they were incubated 95 °C for 90 min, centrifuged at 12,000 rpm for 1 min and the supernatant was recovered. 500 μL of chloroform isoamyl alcohol (24:1) were added, mixed by inversion and centrifuged at 12,000 rpm for 10 min; the supernatant was recovered and 500 μL of cold isopropanol was added. The samples were incubated overnight at -20 °C, then centrifuged at 12,000 rpm for 15 min, decanted and the pellet obtained was washed with 1 mL of ethanol 70 % by centrifuging at 12,000 rpm for 5 min. The pellet was resuspended in 100 μL of nuclease-free sterile water.
X. fastidiosa detection in plants and insects
The bacterium detection was carried out by PCR using the primers RST31 (5’-GCGTTAATTTTCGAAGTGATTCGATTGC-3’) and RST33 (5’-CACCATTCGTATCCCGGTG-3’) (Minsavage et al., 1994). The procedure for PCR reaction consisted of 1 cycle at 95 °C for 5 min, 35 cycles of amplification: 95 °C for 30 s, 60 ° C for 30 s, 72 ° C for 45 s and the final extension at 72 ºC for 7 minutes. The amplified products were analyzed by electrophoresis in a 1.5 % agarose gel, ethidium bromide was used for staining.
X. fastidiosa subsp. multiplex detection
The samples that were positive for X. fastidiosa were again analyzed by PCR for the subspecies identification using the specific primers for the subsp. multiplex XF1968-L (5’-GGAGGTTTACCGAAGACA GAT-3’) and XF1968-R (5’ATCCACAGTAAAACCACATGC-3’), ALM1 (5’CTGCAG AATTGGAAACTTCAG-3’) and ALM2 (5’-GCCACACGTGATCTATGAA-3’) (Hernández-Martínez et al., 2006). The amplification consisted of 1 cycle at 94 °C for 5 min, 40 cycles at 94 °C for 1 min, annealing temperature 55 °C for 1 min, 72 °C for 1 min and the final extension was one cycle at 72 °C for 10 min. The amplified products were analyzed by electrophoresis in a 1.5 % agarose gel.
Results
Insects identification and X. fastidiosa detection in insects
Six genera and four species of leafhoppers were identified through morphological observations and with the use taxonomic keys. One of the leafhoppers was identified as Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae: Cicadellinae) commonly known as glassy winged sharpshoot (Figure 1). Of the genera analyzed by PCR, only X. fastidiosa was detected in H. vitripennis (Table 1), which is reported as the main vector of the bacterium. In relation to the above, Almeida & Purcell (2003) showed that H. vitripennis is a X. fastidiosa vector through a greenhouse transmission experiment. Likewise, Hopkins & Purcell (2002) confirmed that H. vitripennis is the main vector transmitter of bacterium when relating the coincidence of patterns of high insects populations with the appearance of new outbreaks of Pierce’s disease in Napa Valley, Sonoma and Temecula in United States. The literature does not mention other genera of leafhoppers identified in this research as insects vectors of X. fastidiosa.
X. fastidiosa detection in plants
Of the 22 plants species analyzed, X. fastidiosa was detected in vine plants (samples vine 1 and wild vine), in apricot (samples 1, 2 and 3) and in ash (samples 1 and 2) (Table 2). The identification was made by observing a band of 733 bp in electrophoresis gel. The results found in this study are similar to those described in the database of European Food Safety Authority (EFSA) in 2016, which indicate that X. fastidiosa subsp. fastidiosa was detected in vine plants, while X. fastidiosa subsp. multiplex is present in ash and apricot trees, as well as in olive trees.
Table 2 PCR test results in plants for X. fastidiosa detection.
| Plant | Scientific name | PCR Result |
|---|---|---|
| Wild vine | Vitis sp. | Positive |
| Eucalyptus | Eucalyptus sp. | Negative |
| Rose | Rosa sp. | Negative |
| Pear | Pyrus communis | Negative |
| Peach | Prunus domestica | Negative |
| Oriental persimmon | Diospyrus kaki | Negative |
| Carnation | Dianthus caryophyllus | Negative |
| Chinese palm | Yucca filifera | Negative |
| Pecan | Carya illinoensis | Negative |
| Vine 1 | Vitis vinifera | Positive |
| Vine 2 | Vitis vinifera | Negative |
| Jupiter tree | Lagerstroemia indica | Negative |
| Cedar | Cedrela sp. | Negative |
| Japanese privet | Ligustrum japonicum | Negative |
| Olive | Olea europea | Negative |
| Lily | Lilium candidum | Negative |
| Johnson grass | Sorghum alepense | Negative |
| Apricot 1 | Prunus armeniaca | Positive |
| Apricot 2 | Prunus armeniaca | Positive |
| Apricot 3 | Prunus armeniaca | Positive |
| Mandarin | Citrus reticulata | Negative |
| Bouganvilla | Bougainvillea sp. | Negative |
| Avocado | Persea americana | Negative |
| Fig | Ficus carica | Negative |
| Ash 1 | Fraxynus sp. | Positive |
| Ash 2 | Fraxynus sp. | Positive |
| Ash 3 | Fraxynus sp. | Negative |
| Ash 4 | Fraxynus sp. | Negative |
X. fastidiosa subsp. multiplex detection
The identification of X. fastidiosa subsp. multiplex was carried out using the primers XF1968-L / XF1968-R and ALM1/ALM2. The bacterium was detected in apricot plants (samples 1, 2 and 3) and ash (samples 1 and 2) by observing an amplification of 638 and 521 bp corresponding to the aforementioned primers. The PCR multiprimers test with primers pairs: XF1968-L/ XF1968-R, XF2542-L/XF2542-R and ALM1/ALM2 used to differentiate X. fastidiosa in infected vine, almond and flower laurel plants was developed by Hernández-Martínez et al. (2006). In their assay, the subspecies were differentiated based on the set of primers they amplified; based on this, they reported that the samples they amplified with the three pairs of primers correspond to X. fastidiosa subsp. multiplex.
Conclusions
It was shown that X. fastidiosa is affecting different hosts in wine-growing area in Parras, Coahuila. In addition to detecting X. fastidiosa in commercial vine plants and wild grapevine, X. fastidiosa subsp. mutiplex in other hosts such as ash and apricot was identified. Likewise, X. fastidiosa was detected in H. vitripennis which is a potential vector of the bacterium in Parras Valley, Coahuila, Mexico.
