Materials and methods

Results

During the sampling no plants with typical symptoms of pineapple mealybug wilt described or shown in photographs in different publications were observed. The most frequently observed alterations were flaccidity, chlorosis, and reddish coloration in the center of leaves (Figure 1). No mealybugs were observed in any of the collected plants as well as no visible damage or necrosis in the roots were recorded in those showing wilting.

Figure 1 Pineapple plants grown in the region of El Bajo Papaloapan with putative symptoms of mealybug wilt: A: apex leaf necrosis, chlorosis and reddish coloration; B: reddish coloration in the central part of the leaves and downward curvature of the apex; C: Asymptomatic plant; D: flaccidity and apical necrosis of leaves; E: flaccidity, severe chlorosis and reddening of the central part of the leaves; F: yellow chlorotic spots, some with necrotic center; G: Asymptomatic plant; H: wilting, chlorosis in leaves of the middle part and reddening of upper leaves; I: Asymptomatic plant; J: reduction of growth and irregular chlorotic spots; K: chlorosis and reddening of lower leaves; L: reduction of growth, chlorosis of upper leaves, reddening and flaccidity of lower leaves. Plants D and E were of MD2, the rest of Cayena. 

PMWaV-1 was found in one sample (called as PMWaV-1Mexico) showing reduction of growth and irregular chlorotic spots (Figure 1J) and PMWaV-3 was present in one sample too (called as PMWaV-3Mexico) with chlorosis and reddening of lower leaves (Figure 1K), both collected at Isla, Veracruz. PMWaV-2 was not detected in any of the samples analyzed in this study. Both genomic fragments of PMWaV-1Mexico (GenBank access KC800714.1) and PMWaV-3Mexico (GenBank access KC800715.1) sequenced in this work are located within the hsp70 gene, which encodes the heat shock 70-like protein into the ORF 3. PMWaV-1 sequence (589pb) corresponded to the 8.386 8.974 positions of the genome of a Hawaiian PMWaV-1 isolate (GenBank accession AF414119), and PMWaV-3 sequence (324pb) corresponded to the 7.004 7.327 positions of the genome of a Hawaiian PMWaV-3 isolate (GenBank accession DQ399259.2).

The isolate PMWaV-1Mexico showed a 100 % of identity with Cuban isolate and 98 % higher than the rest of isolates, except with Australian isolate (88.6 % of identity). By the other hand, the isolate PMWaV-3Mexico showed a 95.5 % identity higher with the rest of isolates in its group, except with Thailand isolate (88.2 % of identity). Figure 2 shows the similarity of Mexican isolates in relation with those reported in GenBank used for comparison. PMWaV-1Mexico and PMWaV3Mexico grouped with groups 1 and 3, respectively, but no with groups 2, 4 and 5.

Figure 2 Neighbor-Joining tree of 17 PMWaV isolates based on partial sequences of hsp70 gene. Sequences align of isolates PMWaV-1Mexico and PMWaV-3Mexico with sequences of all PMWaV groups found in Genbank. 

Discussion

Mealybug wilt of pineapple (MWP) is a disease characterized by a severe dieback, reddening of leaves, wilt and collapse of mature plants associated with the viruses Pineapple mealybug wilt associated virus-1 (PMWaV-1), and Pineapple mealybug wilt associated virus-3 (PMWaV-3) (^{Sether and Hu, 2000}). In this study it was not observed in any of the plants analyzed the typical symptoms described in literature for MWP. PMWaV-1 was detected in a plant showing reduction of growth and irregular chlorotic spots on all leaves, especially the mature ones (Figure 1), these symptoms are different from those observed in field by ^{Hu et al., (1997)} who also mentioned that a high percentage of plants with MWP are infected with PMWaV-1 while others are asymptomatic. PMWaV-1 is limited to phloem; it is not transmitted mechanically and in field is spread by the mealybug Dysmicoccus brevipes and D. neobrevipes (^{Sether et al., 1998}). In this study did not find mealybugs in any of the sampled plants including the one that tested positive for PMWaV-1, however, this insect is a common pest in the area of study and this form of transmission in field is not discarded; it is possible that its spread may also occur in a vegetative form too (^{Sether et al., 2005}) since this virus causes asymptomatic infections (^{Hu et al., 1997}). In the area of study a reduction of the crop yield has not been observed; however, the presence of PMWaV-1 in the region can eventually affect the production if vector management and water supply is neglected since it has been shown that infection by this virus and limited irrigation produce a negative effect (6.7 and 4.2 %, respectively) and an additive effect (13.4 %) on size and weight of the pineapple fruit (^{Sether and Hu, 2001}). The isolate of PMWaV-1 detected in this study is similar to that reported in Cuba (Figure 2) due perhaps to the highest trade exchange of propagative material between the two countries and least similar to isolates from Australia and Hawaii related to differences in systems production, cultivated plant material and prevailing environmental conditions in all three cases (^{Sether et al., 2010}).

Like PMWaV-1, PMWaV-3 does not induce MWP by itself or together with the mealybug feeding in Hawaii (^{Sether et al., 2005}) and it is unknown what is its effect on the production of pineapple (^{Sether et al., 2009}). Only one plant from the same location that showed different symptoms resulted positive for PMWaV-3. This plant had a reddish coloration in the central part of the mature leaves while young ones had a dark green, and in general did not show a decrease in growth nor flaccidity. As already mentioned, mealybug wilt of pineapple (MWP) is one of the most devastating diseases of this crop worldwide and has been associated with five different ampeloviruses (designated as Pineapple mealybug wilt associated virus-1 to 5) (^{Gambley et al., 2008}). However, it has been shown that the typical symptoms of this disease are only produced when infection simultaneously occurs of Pineapple mealybug wilt associated virus-2 and feeding of Dysmicoccus brevipes and/or D. neobrevipes (^{Sether et al., 1998}). In this study mealybugs were not observed and PMWaV-2 was no detected in the samples analyzed, which is similar to that reported in Hawaii where the incidence of this virus ranges from 0 to 20 % depending on the production system and the hybrid used (^{Sether et al., 2001}) and can cause up to 100 % losses of the fruits when it is present (^{Sether and Hu, 2001}). However, it is necessary to analyze a larger number of samples to know the situation of PMWaV-2 in the area of study. Since PMWaV-1Mexico and PMWaV-3Mexico are more distant and less similar to the Australian and Thailand isolates, respectively, it could suggest that these isolates had different geographical origins. However, it would be necessary to study other genes and largest number of isolates to confirm this hypothesis.

Conclusions

Pineapple mealybug wilt associated virus-1 (PMWaV-1) was detected in a pineapple plant showing reduction of growth and irregular chlorotic leaf spots and Pineapple mealybug wilt associated virus-3 (PMWaV-3) in one plant showed a reddish coloration in the center of mature leaves. The PMWaV-1Mexico isolate is similar to one reported in Cuba and somewhat different from that of Australia and Hawaii, while PMWaV-3Mexico isolate show genetic differences with other isolates of PMWaV-3 reported in the Genbank.