Pineapple mealybug wilt-associated viruses 1, 2, and 3 are associated with mealybug wilt disease of pineapple in Peru

Mealybug wilt of pineapple (MWP) is the most important disease of pineapple worldwide. During the months of September and November 2020, pineapple production fields of the cultivar Hawaiiana and hybrid MD-2 in Satipo and Chanchamayo provinces of Peru, showed typical symptoms of MWP. Based on symptoms, the disease incidence level ranged between 70 and 90%. Symptomatic and asymptomatic foliar samples were collected and tested by RT-PCR using primers specific for pineapple mealybug wilt virus (PMWaV) 1, 2, and 3. Selected amplified fragments were sequenced and analyzed with bioinformatics tools. PMWaV-1, 2, and 3 were detected in symptomatic samples of both cultivars but not in asymptomatic samples. PMWaV-1 showed a nucleotide identity of 91.70% with PMWaV-1 from Thailand; PMWaV-2, 99.81% with PMWaV-2 from Thailand; and PMWaV-3, 99.09% with PMWaV-3 from Cuba. Mixed infections of PMWaV-1, 2, and 3 were also detected in both cultivars. To our knowledge, this is the first report of PMWaV-1, 2, and 3, associated with pineapple in Peru.

Mealybug wilt of pineapple (MWP) disease is the most important disease of pineapple worldwide (Sether et al. 2010). A complex of three viruses has been associated with this disease, and virus nomenclature was based on the symptoms of the disease and the type of insect vectors. As a result, the names pineapple mealybug wiltassociated virus (PMWaV) 1, 2, and 3 have been proposed for the viruses (Sether et al. 2005). The three species have been recognized by the International Committee on Taxonomy of Viruses and placed in the genus Ampelovirus, family Closteroviridae (Gambley et al. 2008). The size of the genomes of PMWaV-1, PMWaV-2, and PMWaV-3 are 13,071 bp, 16,259 bp, and 13,298 bp respectively (Green et al. 2020). The genome of PMWaV-1, 2, and 3 consists of single-stranded, linear, positive-sense RNA containing up to 10 open reading frames (ORF). The ORF1a encodes a protease, a methyltransferase, and a helicase; ORF1b an RNAdependent RNA polymerase; ORF2 a hydrophobic protein; ORF3 a heat shock protein 70 homolog; ORF4 a protein of unknown function; ORF5 a capsid protein; and ORF6 the capsid protein duplicate (Sether et al. 2010). Additionally, Pineapple (Ananas comosus L.) is a monocotyledonous perennial plant species in the family Bromeliaceae tolerant to water scarcity (Krauss 1948). According to the Food and Agriculture Association, the top five pineapple-producing countries are Costa Rica, the Philippines, Brazil, Thailand, and Indonesia. In 2018, Peru reported a harvested area of 15,901 ha, with a yield of 344,925 kg/ha, and a production of 548,465 tons of pineapple (FAOSTAT 2020). According to the Ministry of Agrarian Development and Irrigation, during 2019, the regions with the highest production were Junin (436,768 tons), La Libertad (22,903 tons), Puno PMWaV-2 contains ORF7, ORF8, and ORF9 (proteins of unknown function) (Green et al. 2020). These viruses cause symptoms of browning and reddening of leaves, reduction of root mass, wilting, and leaf necrosis that eventually lead to plant death (Sether and Hu 2002).
During the production season of 2020 (September to November), pineapple fields of the cultivar Hawaiiana and the hybrid MD-2 in the provinces of Satipo and Chanchamayo (Junín region) were inspected and symptoms of reddening and chlorosis of leaves, regressive apical necrosis, and reduced growth and root mass were observed. The symptoms were similar to those associated with MWP disease, which was reported to cause a decrease in the yield of these two cultivars (Hernandez-Rodriguez et al. 2014). Therefore, the objective of this investigation was to determine if the pineapple mealybug wilt-associated viruses were associated with the disease symptoms on pineapple in the provinces of Satipo and Chanchamayo (Junín region) in Peru.
In 2020, two field samplings were conducted, one in September, which consisted of 10 samples of the cultivar Hawaiiana from the locality of San Ramon de Pangoa (Satipo province). The second sampling was conducted in November and consisted of 48 samples of hybrid MD-2 collected from the localities of Celendin, Mazamari, and Alto Belen and 30 samples of the cultivar Hawaiiana collected from San Ramon de Pangoa (Satipo province) and Alto Pichanaki (Chanchamayo province) of the department of Junín (Table 1). Samples consisted of leaf tissue from plants exhibiting symptoms similar to those reported for MWP. Additionally, symptomless samples were collected. Plants of the cultivar Hawaiiana exhibited symptoms of yellowing, reddening, apical necrosis, and dwarfing (Fig. 1b). The hybrid MD-2 showed yellowing, apical necrosis, chlorotic halos, and dwarfing (Fig. 1c). The percentage of disease incidence was estimated visually in the field, using the W sampling method of Gottwald (1995). To conduct RT-PCR, total RNA was extracted using TRIzol (Invitrogen™). The quality of the RNA was estimated using a 1% agarose gel and quantified with a NanoDrop™ spectrophotometer (ThermoSci-entific™). Subsequently, the Maxima H Minus First Strand cDNA Synthesis Kit (ThermoScientific™) was used to obtain the cDNA. Specific primers for PMWaV-1 [225 (forward 5′-ACAGGAAGGACAACACACTCAC-3′) and 226 (reverse 5′-CGCACAAACTTCAAGCAAGCAATC-3′)], PMWaV-2 [224 (forward 5′-CATACACGAACTAGACT-CATACACG-3′) and 223 (reverse 5′-CCATCCACAC-CAATTTTACTAC-3′)]; and PMWaV-3 [264 (forward 5′-AGTTCACTGTAGATTTCGGA-3′) and 263 (reverse 5′-ATTGATGGATGGATGTGTATCG-3′)] were used, which amplify 590, 610 and 490 bp long fragments of the Hsp70 gene of PMWaV-1, 2 and 3, respectively. The amplification parameters were as reported by Sether et al. (2005) and Hernandez et al. (2010). A total of 19 PCR-positive samples (5 for PMWaV-1 and 7 each for PMWaV-2 and PMWaV-3) from San Ramon de Pangoa, Celedin and Mazamari (cultivar Hawaiiana and hybrid MD-2), respectively, were purified using the DNA Fragments Extraction Gel/ PCR Kit (Geneaid®) and sequenced by the Sanger method at Macrogen (South Korea) in both directions using the corresponding primers for each of the three viruses. The nucleotide sequences of the isolates were submitted to BLASTn (Altschul et al. 1990). Related sequences were downloaded from the GenBank and used for multiple alignments with the ClustalW program (Thompson et al. 1994). To establish phylogenetic relationships, a tree was constructed using MEGA 6 (Tamura et al. 2013) with 1000 replicates to establish the bootstrap value.
In the fields where samples were collected, the presence of mealybugs was observed (Fig. 1d). Mealybugs were collected and identified as Dysmicoccus brevipes by PCR and sequencing of the internal transcribed spacer (ITS) region 2 (Malausa et al. 2011). The estimated disease incidence at the field level was between 70 − 90% (Table 1). In the localities of Celendín, Mazamari, and Alto Belén, plants of the hybrid MD-2 had a disease incidence of 70%, 90%, and 90%, respectively; while in San Ramón de Pangoa and Alto Pichanaki, plants of the cultivar Hawaiiana had an incidence of 90% (Fig. 1a). As shown in Fig. 2a-c, PMWaV-1, PMWaV-2, and PMWaV-3 were detected in Celendín, Mazamari, Alto Belén (hybrid MD-2), and San Ramón de Pangoa (cultivar Hawaiiana); while only PMWaV-3 was detected in Alto Pichanaki (cultivar Hawaiiana). The viruses were not detected in asymptomatic samples. In the samples collected in the province of Satipo (cultivar Hawaiiana), mixed infections of PMWaV-1, 2, and 3 were detected in two samples.
Likewise, mixed infections in the hybrid MD-2 of the three viruses were detected in five samples (Fig. 2a, b, and c).
In Latin America, PMWaV-1, 2, and 3 have been reported infecting pineapple in Cuba (Hernandez-Rodriguez et al. 2014), PMWaV-1 in Ecuador (Alvarez et al. 2015), and PMWaV-1 and 3 in Mexico (Ochoa-Martínez et al. 2016). Based on symptoms, presence of the insect vector, RT-PCR tests, and nucleotide sequencing of selected RT-PCR products, we have identified PMWaV-1, 2, and 3 infecting pineapples in Peru. The Peruvian isolate of PMWaV-1 appears to be a strain of PMWaV-1 reported in other countries (Ochoa-Martínez et al. 2016;Hernandez-Rodríguez et al. 2017;Nyarko et al. 2019) while Peruvian isolates of PMWaV-2 and 3 were essentially identical to corresponding isolates reported in Thailand and Cuba, respectively (Hernandez-Rodriguez et al. 2014). In Cuba, mixed infections of the three viruses have been previously reported in the pineapple in the hybrid MD-2 (Hernandez-Rodríguez et al. 2017). However, to our knowledge, this is the first report of mixed infection of PMWaV-1, 2, and 3 in cultivar Hawaiiana worldwide.
Although the production fields in the localities of Celendin, Mazamari, Alto Belen, San Ramon de Pangoa, and Alto Pichanaki showed symptoms of MWP disease, the pineapple growers were unaware of the etiology of this disease and applied unsuccessful control strategies. These infected to our knowledge, this is the first report of PMWaV-1, 2, and 3 associated with pineapple in Peru.
fields are likely sources of inoculum for neighboring fields. PMWaV-1, 2, and 3 have been reported to be associated with the presence of mealybugs in the genus Dysmicoccus (Gambley et al. 2008;Sether et al. 2010). The high incidence of these viruses (70-90%) observed in the cultivar Hawaiiana and hybrid MD-2 in production fields in the provinces of Chanchamayo and Satipo is probably due to both the presence of the insect vector and the vegetative propagation practices applied for this crop. In these provinces, to plant a new crop field, growers obtain vegetative seed (tillers) from surrounding fields. Therefore, spread of the viruses occurs if tillers are collected from infected plants (Solovyev et al. 2014).
The spread of PMWaV-1, 2, and 3 represents a threat to pineapple production (Amari et al. 2021). To develop appropriate and efficient control strategies, it is necessary to identify the viruses and vectors involved in the MWP disease complex. Furthermore, it is necessary to implement an in vitro propagation system to obtain virus-free vegetative seed as reported by Rodríguez et al. (2016). In conclusion, Fig. 2 Results of RT-PCR amplification of the Hsp70 gene of pineapple mealybug wilt associated virus (PMWaV) 1, 2, and 3 from pineapple foliar samples. Fragments of approximately 590, 610, and 490 bp were amplified with virus-specific primers for PMWaV-1 (a); PMWaV-2 (b); and PMWaV-3 (c), respectively. In a), b) and c) wells 1-10, samples from San Ramon de Pangoa cultivar Hawaiiana; 11-12, samples from Celendin hybrid MD-2; 13-14, samples from Mazamari hybrid MD-2; 15, sample from Alto Belen hybrid MD-2; 16-17, sample from Alto Pichanaki cultivar Hawaiiana; 18, negative control; and 19, positive control. M, GeneRuler 1 Kb DNA Ladder (ThermoScientific™) Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright