Pineapple Mealybug Wilt-associated Virus Research Articles

Characterization of Six Ampeloviruses Infecting Pineapple in Reunion Island Using a Combination of High-Throughput Sequencing Approaches.

The cultivation of pineapple (Ananas comosus) is threatened worldwide by mealybug wilt disease of pineapple (MWP), whose etiology is not yet fully elucidated. In this study, we characterized pineapple mealybug wilt-associated ampeloviruses (PMWaVs, family Closteroviridae) from a diseased pineapple plant collected from Reunion Island, using a high-throughput sequencing approach combining Illumina short reads and Nanopore long reads. Reads co-assembly resulted in complete or near-complete genomes for six distinct ampeloviruses, including the first complete genome of pineapple mealybug wilt-associated virus 5 (PMWaV5) and that of a new species tentatively named pineapple mealybug wilt-associated virus 7 (PMWaV7). Short reads data provided high genome coverage and sequencing depths for all six viral genomes, contrary to long reads data. The 5' and 3' ends of the genome for most of the six ampeloviruses could be recovered from long reads, providing an alternative to RACE-PCRs. Phylogenetic analyses did not unveil any geographic structuring of the diversity of PMWaV1, PMWaV2 and PMWaV3 isolates, supporting the current hypothesis that PMWaVs were mainly spread by human activity and vegetative propagation.

Economically Imperative Ananas comosus Diseases, Status, and Its Control Measures Documented in Producing Countries

Ananas comosus, commonly known as pineapple, is a fruit with a large potential market as a commodity and commercial fruit. Numerous pests and diseases affect pineapple, directly or indirectly, by lowering the quality and quantity. The fungal causative agents, namely Fusarium ananatum and Thalaromyces stolii (previously named Penicillium funiculosum), cause fruitlet core rot (FCR) and fusariosis by Fusarium guttiforme. Bacteria heart rot (BHR) is an infection by Erwinia chrysanthemi, newly known as Dickeya zeae. Nevertheless, the mealybug wilt of pineapple (MWP) is another pineapple treat to susceptible pineapple varieties caused by pineapple mealybug wilt-associated viruses (PMWaVs). Other diseases include destruction caused by pathogenic nematodes. This review discusses the status of these diseases and the control measures that greatly affect the economy of pineapple-producing countries due to the economic significance of these crops. Growers need up-to-date information on the identity of the diseases that affect pineapple crops in the various countries that produce them to effectively manage the diseases in the field.

Ecologically Based Management of Pineapple Mealybug Wilt: Controlling Dysmicoccus brevipes Mealybug Populations with Salicylic Acid Analogs and Plant Extracts

Mealybug wilt of pineapple (MWP) is a destructive disease worldwide caused by a parasitic complex that includes Pineapple Mealybug Wilt-associated Viruses (PMWaVs) and mealybugs (Dysmicoccus brevipes), which concurrently act as vectors for these viruses. Reducing the mealybug population is key to managing MWP, which is achieved in intensive production systems through the use of insecticides. SA (salicylic acid), ASM (acibenzolar-S-methyl), BABA (β-aminobutyric acid), and MeSA (methyl salicylate) are key components of systemic acquired resistance (SAR), the defense mechanism of plants against biotrophic agents such as mealybugs. In this study, these compounds were applied either as pure chemicals and/or as a major constituent of plant extracts. Both the Hawaiian hybrid MD-2 and Queen Victoria tissue culture plants, as well as suckers used for vegetative propagation, were treated with these compounds by direct application on the soil of pineapple pots. Subsequently, five mealybugs were released on each plant or each daughter plant in case of a transgenerational experiment; then, after 45 days, the number of mealybugs was counted. Exogenous SA, ASM, and MeSA reduced the population of mealybugs by a minimum of 50% and up to 80%. These SAR-inducing treatments could be an interesting alternative for controlling mealybugs and are already used in other pathosystems. The SAR mechanisms behind this effect are yet to be confirmed by molecular and enzymatic markers. ASM and MeSA are promising treatments for pineapples using tissue culture plants or traditional shoots.

The vector of Pineapple Mealybug Wilt-associated Virus (PMWaV) in Sipahutar pineapples in North Tapanuli, Indonesia

Abstract. Lisnawita, Tantawi AR, Tobing MC, Hutahayan AJ, Saragih WS, Sartiami D. 2023. The vector of Pineapple Mealybug Wilt-associated Virus (PMWaV) in Sipahutar pineapples in North Tapanuli, Indonesia. Biodiversitas 24: 4052-4059. The Pineapple Mealybug Wilt-associated Virus (PMWaV) disease is one of the most important diseases in pineapples. It Is widely spread in almost all pineapple plantations, including those in North Tapanuli District, North Sumatra Province, Indonesia. Currently, there is still little information on the PMWaV disease and the species of mealybug found in Sipahutar pineapples in the North Tapanuli District. This research aims to identify mealybugs as the vectors that carry the virus that causes PMWaV disease in Sipahutar pineapples. Mealybug samples were taken from 12 villages in four sub-districts in the center of Sipahutar pineapples plantations in the North Tapanuli District. The four sub-districts were Sipahutar Sub-district from Siabal Abal IV Village, Sabungan Nihuta I Village, and Sabungan Nihuta II Village; Siborongborong Sub-district from Bahal Batu I Village, Bahal Batu II Village, and Lobu Siregar Village; Tarutung Sub-district from Parbaju Tonga Village, Jambur Nauli Village, and Sihujur Village; Pangaribuan Sub-district from Pansurnatolu Village, Sigotom Village, and Rahut Bosi Village. Samples were identified morphologically and molecularly. The results of the identification using both methods showed that the mealybugs found on Sipahutar pineapples with PMWaV symptoms from the 12 villages were Dysmicoccus brevipes species.

Phytonematode on Pineapple (Ananas comosus L. Merrill) GP3 Clone With Symptoms of Pineapple mealybug wilt-associated virus (PMWaV)

Pineapple (Ananas comosus L. Merr) is one of Indonesia’s important fruit commodities. Indonesia became the world’s fourth-largest pineapple producer in 2020. The monoculture cultivation system can lead to the continuous presence of pests and diseases in plantation areas. Mealybug wilt disease is caused by Pineapple mealybug wilt-associated virus (PMWaV) and its thought to be associated with phytomatodes. This study aims to determine the type of phytomatodes in pineapple plants with three health degrees: healthy plants, moderate symptoms, and severe symptoms. The soil and roots were sampled at 3, 5, 13, and 14 MAP (Month after Planting). Nematodes were extracted from the soil using the flotation-centrifugation method, while the root samples were extracted using the mist chamber method. Identification of nematode morphology was based on anterior (mouth and stylet shape), posterior (tail shape and reproductive organs), and the type of annulation or nematode cuticle surface. Seven genera of phytomatodes were identified from soil and root samples: Pratylenchus spp., Rotylenchulus spp., Meloidogyne spp., Helicotylenchus spp., Crinonemoides spp., Tylenchus spp., and Rotylenchus spp.. The dominant phytonematodes in the soil samples were Rotylenchulus spp. (66.67/100 ml of soil), while Pratylenchus spp. predominated in the roots (58.33/10 g of roots). Based on the three degrees of plant health, the phytonematodes that dominated the soil samples were Rotylenchulus spp. on the health degree of healthy and moderate symptoms, and Meloidogyne spp. dominate the sample at severe symptoms. Pratylenchus spp. dominated the root samples at healthy and severe symptoms, while Rotylenchulus spp. predominate in moderate symptomatic samples.

Detection and identification of viruses associated to mealybug wilt pineapple in Blitar, East Java, Indonesia

Pineapple is one of the essential export commodities in Indonesia. However, its production is constrained by pathogen infections, including viruses. Recently, a field surveyed at a pineapple production area in Blitar regency found virus-like symptoms such as leaf reddish intermingle with chlorosis and necrotic leaf tips with incidence up to 50% on clone 7350. However, until present, the genetic identity of causal viruses has been limitedly available in Indonesia. The study aimed to detect and identify the virus species associated with mealybug wilt pineapple (MWP) in Blitar regency. Total RNA was extracted and detected by one-step RT-PCR using a universal primer of Ampelovirus, a specific primer of pineapple mealybug wilt-associated virus (PMWaV) 1 and 2, and confirmed by DNA sequencing. The PMWaV-1 and PMWaV-2 were successfully amplified by specific primers, while by Ampelovirus universal primers negatively amplified any DNA. It confirmed that the causal virus is associated with corresponding symptoms only by PMWaV-1 and -2 on clone 7350. Based on partial sequences of the HSP70 gene confirmed the existence of both species. The identity of PMWaV-1 Blitar isolates showed the highest similarity of nucleotide and amino acid to isolates from Taiwan, Mexico, and Ghana. The PMWaV-2 Blitar isolates showed the highest similarity to isolates from Taiwan, Mauritius, Cuba, and Ghana. The highly incidence of MWP suggests that cultivating virus-free vegetative propagation will become the best strategy to manage the viruses in the fields.

Occurrence and distribution of pineapple mealybug wilt-associated viruses (PMWaVs) in MD2 pineapple fields in the Valle del Cauca Department, Colombia

MD2 pineapple production in the Valle del Cauca Department, Colombia, has increased in recent years due to its preference in the national and international markets. Pineapple mealybug wilt (PMW), reported in all production areas around the world, is associated with two mealybug species, Dysmicoccus brevipes and D. neobrevipes, and a group of virus species known as pineapple mealybug wilt-associated viruses (PMWaVs). In a previous study, D. brevipes was determined to be the main mealybug species associated with pineapples in the region; hence, the objective of this study was to determine the occurrence and distribution of PMWaVs in pineapple plants and specimens of D. brevipes collected in five MD2 pineapple fields in the Department of Valle del Cauca. Our results confirmed the presence of three virus species in both plant and mealybug specimens. PMWaV3 showed the highest detection frequencies (92.6% and 88.7%), followed by PMWaV1 (26.5% and 8.3%) and PMWaV2 (12.0% and 2.0%) in plant and mealybug samples, respectively. Mixed infections of PMWaVs were found also in the studied plant material. The most common virus association consisting of PMWaV1 and PMWaV3 was found in 30 plant samples (20%). The correlation analysis between each virus species found in mealybugs and their pineapple host suggests that PMWaV3 is not only transmitted through infested plant material, but that D. brevipes may also play a role in the dynamics of disease dissemination. The results of this study contribute to the understanding of PMWaV transmission in MD2 pineapple-cultivated areas in the Valle del Cauca region.

MANAGEMENT OF WEEDS ON PINEAPPLE FARM USING SELECTIVE HERBICIDE ON PEAT SOIL

Weeds pose problem to the production of pineapple by competing for nutrients, space, light and water. Pineapple production without good weed management could reduce yield and could cause insects infestation such as mealybugs that could spread serious disease for instance pineapple mealybug wilt-associated virus. Weed management using selective herbicide kills the weed without affecting pineapple plant growth and fruit quality. Four different selective herbicides were chosen with active ingredients of Atrazine, Ametryn, Bromacil and Diuron. Three frequencies of application were chosen which are 8, 10 or 12 weeks. The results of plant growth rate showed no significant different between treatment except on unweeded plot. Bromacil showed the best control on weed followed by Ametryn, Diuron and Atrazine over period of time. Eight weeks frequency of application did not differ to 10 weeks but both frequencies provide better weeds management over 12 weeks frequency of application. Major weeds species recorded were Melastoma malabathricum, Asystasia gangetica and Fimbristylis pauciflora. The used of selective herbicide is suitable in controlling weeds on pineapple plantation on peat soil without affecting plant growth rate.

Genetic Diversity of Viral Populations Associated with Ananas Germplasm and Improvement of Virus Diagnostic Protocols.

Pineapple (Ananas comosus L. [Merr.]) accessions from the U.S. Tropical Plant Genetic Resources and Disease Research (TPGRDR) in Hilo, Hawaii were subjected to RNA-sequencing to study the occurrence of viral populations associated with this vegetatively propagated crop. Analysis of high-throughput sequencing data obtained from 24 germplasm accessions and public domain transcriptome shotgun assembly (TSA) data identified two novel sadwaviruses, putatively named "pineapple secovirus C" (PSV-C) and "pineapple secovirus D" (PSV-D). They shared low amino acid sequence identity (from 34.8 to 41.3%) compared with their homologs in the Pro-pol region of the previously reported PSV-A and PSV-B. The complete genome (7485 bp) corresponding to a previously reported partial sequence of the badnavirus, pineapple bacilliform ER virus (PBERV), was retrieved from one of the datasets. Overall, we discovered a total of 69 viral sequences representing ten members within the Ampelovirus, Sadwavirus, and Badnavirus genera. Genetic diversity and recombination events were found in members of the pineapple mealybug wilt-associated virus (PMWaV) complex as well as PSVs. PMWaV-1, -3, and -6 presented recombination events across the quintuple gene block, while no recombination events were found for PMWaV-2. High recombination frequency of the RNA1 and RNA2 molecules from PSV-A and PSV-B were congruent with the diversity found by phylogenetic analyses. Here, we also report the development and improvement of RT-PCR diagnostic protocols for the specific identification and detection of viruses infecting pineapple based on the diverse viral populations characterized in this study. Given the high occurrence of recombination events, diversity, and discovery of viruses found in Ananas germplasm, the reported and validated RT-PCR assays represent an important advance for surveillance of viral infections of pineapple.

Survey and distribution of Pineapple Wilt Mealybug Wilt associated Virus (PMWaV) on pineapple plants in North Tapanuli, Indonesia

The authors conducted a survey of Pineapple Mealybug Wilt-associated Virus (PMWaV) on pineapple plants in four pineapple-producing sub-districts in North Tapanuli, namely Sipahutar, Pangaribuan, Siborongborong, and Tarutung. This survey aims to detect the presence and distribution of PMWaV in North Tapanuli. The survey found PMWaV in pineapple plants in all survey locations with varying symptoms such as wilting to a yellow-red colour and curling leaf tips, curling and drying leaves and leaf tips, and showing symptoms of wilting to a redand yellow colour. The results showed an incidence rate between 15.6 to 80%. Sampang ni Huta V, Sipahutar, showed the lowest incidence rate (15.6%), while Lobu Siregar, Siborongborong, showed the highest (80.0%). Meanwhile, the severity rate was between 14.0 and 65.0%. Pancur Natolu, Pangaribuan, showed the highest severity rate (65.0%, severe), while Siabal-abal II, Sipahutar, showed the lowest (14.0%, moderate).

Pineapple mealybug wilt-associated viruses (pineapple mealybug wilt (PMBW))

Pineapple mealybug wilt-associated viruses (pineapple mealybug wilt (PMBW))

A novel ampelovirus associated with mealybug wilt of pineapple (Ananas comosus).

Mealybug wilt of pineapple (MWP) is the most important and complex viral disease affecting pineapple worldwide. High-throughput sequencing was conducted to characterize a new virus identified only in symptomatic pineapple plants and tentatively named pineapple mealybug wilt-associated virus 6 (PMWaV-6). Data analyses revealed a genome of 17,854 nucleotides with an organization resembling members of the genus Ampelovirus, family Closteroviridae. Encoded proteins shared sequence identity with the corresponding proteins of grapevine leafroll-associated virus 3, blackberry vein banding-associated virus, and PMWaV-2. The present study reports the discovery of PMWaV-6, a putative and distinct new member of the genus Ampelovirus, subgroup I, its potential involvement in MWP, and the development of PMWaV-6-specific RT-PCR assays to detect and monitor this virus in field samples.

Pineapple mealybug wilt-associated virus (PMWaV) on Sipahutar pineapple, in North Tapanuli, Indonesia

Pineapple Mealybug Wilt-associated Virus (PMWaV) is one of the main diseases that affect pineapples all over the world, including Sipahutar, North Tapanuli, Indonesia. Currently, there are no studies or information on PMWaV disease as well as the species of mealybugs found in sipahutar pineapples. Therefore, this study aims to identify the mealybugs causing PMWaV in sipahutar pineapple. Data were obtained from 3 (three) villages, namely Siabal-abal II, Onan Runggu, and Sabunganni Huta V, to assess the disease incidence and collect mealybugs. The results showed that the disease incidence of PMWaV in the 3 villages were between 15.6 - 21.6%. The highest disease incidence was found in Siabal-abal II (21.6%), followed by Onan Runggu (21.3%), and SabunganniHuta V (15.6%). The mealybugs found on pineapple sipahutar with PMWaV symptoms in the 3 surveyed villages were identified as Dysmicoccus brevipes.

Strategies for vegetative propagation and viral cleaning of a miniature ornamental pineapple hybrid

This study assessed and compared different methods for vegetative propagation of a miniature ornamental pineapple hybrid (ORN-MUT), seeking to determine the best method for production of plantlets, as well as for removal of the PMWaV viral complex from plants cultured in vitro, for production of healthy parent plants. Pineapple wilt is a disease that can cause large economic and is caused by a viral complex called Pineapple mealybug wilt-associated virus (PMWaV). For this, four propagation methods were evaluated (conventional, stem sectioning, micropropagation and etiolation of nodal segments). The time necessary for each method and the number of plants formed were assessed. Stem tips (0.5 mm) were cultured and indexed for three PMWaV types. Conventional propagation produced 17 plantlets per plant in 566 days, stem sectioning produced 2.3 plantlets per stem in 591 days, while the conventional micropropagation technique produced 1,284 plants after four subcultures in 778 days. Stems etiolated for 60 days showed peak production in the second subculture, with 1,224 plants. This method required 883 days to obtain plants with ideal size for transplantation to the field. In turn, stems etiolated for 120 days produced 935 plants at the end of four subcultures, with peak output in the third subculture, in which the plants could be cultivated in the field after 943 days. Conventional micropropagation and etiolation for 60 days were the best methods for production of plantlets of the ORN-MUT hybrid. The results of this work showed that the cultivation of shoot tips is an efficient strategy to remove the PMWaV complex and obtain healthy mother plants and can be a useful tool for other varieties of pineapple.

First Report of Three Pineapple Mealybug Wilt-Associated Viruses in Queen Victoria Pineapples in Reunion Island

First Report of Three Pineapple Mealybug Wilt-Associated Viruses in Queen Victoria Pineapples in Reunion Island

Comparison of shoot tip culture and cryotherapy for eradication of ampeloviruses associated with Pineapple mealybug wilt in wild varieties

The Pineapple mealybug wilt-associated virus (PMWaV) provokes a disease that causes considerable losses to pineapple growers due to the reduced leaf tissue turgescence, resulting in leaf necrosis and death in severe cases. In this work, we describe the comparison of shoot tip culture and cryotherapy methods to eradicate the pineapple wilt disease-associated ampeloviruses. Plants from the accessions Ananas comosus var. comosus (AGB-009), var. bracteatus (AGB-119), and var. parguazensis (AGB-376) were indexed by RT-PCR, confirming mixed infections of PMWaV-1, PMWaV-2, and PMWaV-3. The accessions were cultured in vitro and their shoot tips treated by cryotherapy following a droplet vitrification protocol. The regeneration rate from shoot tip culture was 93% for AGB-376 and 100% for the other two accessions. After freezing, AGB-376 had 100% regeneration subsequent to exposure to PVS2 for 45 min, followed by 95% for AGB-009, while for AGB-119 the optimal exposure time was 60 min, with plant regeneration from nearly 80% of the shoot tips. For the accessions AGB-009 and AGB-376, all the recovered plants were virus free by the two methods, while 50% of the plants from accession AGB-119 remained infected. These results indicate that shoot tip culture alone or in association with cryotherapy is a promising routine method for virus removal from pineapple plant tissues and is useful to ensure that backup reserves of pineapple germplasm, conserved by in vitro bank and cryobank, are formed with virus-free plants.

Further genomic characterization of pineapple mealybug wilt-associated viruses using high-throughput sequencing

In this study, we describe the further genomic characterization of several pineapple mealybug wilt-associated virus (PMWaV) members of the genus Ampelovirus, family Closteroviridae, using high-throughput sequencing (HTS). PMWaV-2 is a necessary factor in the complex etiology of mealybug wilt disease of pineapple (MWP). The complete PMWaV-1 genome was described previously, but the genomes of PMWaV-2, PMWaV-3, and putative PMWaV-4 and PMWaV-5 species lack complete characterization. To further study MWP etiology through genomic characterization of PMWaVs, a cDNA library from total RNA of pineapple plants was generated and underwent HTS. A PMWaV-4 genome of 12,932 nucleotides was assembled from the HTS data. Proteins from the PMWaV-4 genome shared high amino acid similarity to those of PMWaV-1. The RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homolog (HSP70h), and coat protein (CP) proteins of PMWaV-4 shared 88, 87, and 85% amino acid identity, respectively, to those of PMWaV-1 (AF414119) indicating PMWaV-4 is a distinct strain of PMWaV-1 but not a distinct ampelovirus species. Therefore, we suggest that PMWaV-4 be designated PMWaV-1 strain 4 (MN539275). Further, we used HTS to obtain a new isolate of PMWaV-1 (MN539276) and supplemented the available sequences of the genomes of PMWaV-2 (MN539272 and MN539273) and PMWaV-3 (MN539274) in their 5′-terminal regions.

The viral complex associated with mealybug wilt disease of pineapple in Cuba

Mealybug wilt of pineapple (MWP) disease has been associated with the infection by a complex of ampeloviruses (Pineapple mealybug wilt-associated virus 1 (PMWaV-1), PMWaV-2, PMWaV-3, PMWaV-4 and PMWaV-5)) (Closteroviridae) and badnaviruses (Pineapple bacilliform CO virus (PBCOV) and Pineapple bacilliform ER virus (PBERV) (Caulimoviridae)). In Cuba, PMWaV-1, PMWaV-2, PMWaV-3 and PBCOV are widespread in commercial pineapple fields, but the association of these viruses with the aetiology of the disease remains unknown. During 2009 to 2012, a survey to detect PMWaV-1, PMWaV-2 and PMWaV-3 by RT-PCR, and PBCOV by non-radioactive Dot Blot hybridization was carried out on samples collected from asymptomatic or MWP symptomatic pineapple plants from 24 commercial fields in 10 provinces and Isla de la Juventud. PMWaV-1, PMWaV-2, PMWaV-3, and PBCOV occurred in simple or mixed infections, whereas PMWaV-2 was the viral species most frequently detected in symptomatic plants. The positive or negative diagnostic results of the RT-PCR to detect PMWaV-2 was more reliably associated with the MWP symptomatic or asymptomatic status of the sampled plants, respectively, than those from PMWaV-1, PMWaV-3 or PBCOV detection techniques. This work discloses the correlation between the infection by PMWaV-2 and the presence of MWP symptoms in Cuba, supporting previous studies indicating the major role of this virus triggering MWP disease.

Bioinformatics approach to the study of the molecular behaviour of mealybug wilt of pineapple

Mealybug wilt of pineapple (MWP) is a disease caused by the Pineapple mealybug wilt-associated virus (PMWaV) complex transmitted by Dysmicoccus brevipes and D. neobrevipes. MWP symptoms are characterized by root dessication, leaf wilting and consequent failure to produce a fruit. The molecular mechanisms involved in the pineapple-PMWaV interaction for MWP symptomatology are still unclear. In this work, mRNAs of asymptomatic and symptomatic pineapple plants were evaluated using Illumina RNA sequencing technology. From a total of 79 million reads per sample, 16,097 genes were identified using STAR aligner and HTseq for paired-end files. Differentially expressed genes (DEGs) between the evaluated groups were estimated using DESeq2 and edgeR, with an FDR cutoff of ≤0.05. A total of 207 DEGs were detected, with 61 upregulated and 146 downregulated in symptomatic plants infected by PMWaV-2. The methodologies improved by the assays presented in this article and the detected DEGs can substantiate further researches with pineapple and the MWP disease.

Conservation, propagation and new paths for pineapple genetic resources

The Embrapa Cassava and Fruits research unit (Embrapa Mandioca e Fruticultura) maintains in its experimental fields a pineapple active germplasm bank (AGB Pineapple) with 700 accessions of the genus Ananas. In recent years, several of these accessions, of all botanical varieties, have been lost as a result of infection by the Pineapple mealybug wilt-associated virus complex (PMWaV). Because of this threat, the establishment of security duplicates is a priority for the managers of AGB Pineapple, to minimize losses in the field as well as to increase the efficiency of preservation. Two strategies to preserve this germplasm have been used in recent years: in vitro conservation and cryopreservation. This work will cover the limitations of the two techniques and in particular the advances achieved, and proposals being analysed for better conservation of this important germplasm. It will also cover the efficiency of propagation techniques, the precautions that need to be considered and their convergence with preservation and genetic improvement of pineapple species. Finally, the creation of a global collaborative structure for genetic resources of the genus Ananas will be discussed, for the purpose of assuring their conservation by different strategies, based on cooperation, to facilitate use of the conservation methods by researchers in several countries.