Metabarcoding of nematode communities associated with Ananas comosus L. (Pineapple)

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Introduction
Pineapple is one of the major fruit crops in the world, including the Philippines. The country ranks third in production worldwide, with an estimated production of 2.86 million metric tons for the past five years (Shahbandeh, 2023). The pineapple plantation area is more than 40,000 hectares, and the concentration of production is in Mindanao and Luzon and is controlled by international companies. The most widely grown cultivars are Queen, Cayenne, Philippine Native or Red Spanish, and the newly developed MD2. In 2019, the country was the second largest pineapple exporter, according to the Food and Agriculture Organization (Hossain, 2016), and was considered the primary export product of the Philippines, same with bananas (Voora et al., 2020). Pineapple production and exportation can be considered at their peak in the Philippines (Balito, 2010). However, problems and challenges might arise and need to be addressed. These include natural calamities, soil degradation, and disease and pest infestation. One of the pests that pose a significant threat to the crop is the plant-parasitic nematodes (Bugnay-Inso and Pedroche, 2021). It has been reported by Garcia et al. (2022) that these roundworms can affect root function, which could lead to crop deterioration and, eventually, death. Thus, it would lead to a substantial decline in yield (approximately 60%). Nematodes are one of the most diverse creatures on earth. Due to its enormity and complexity, the Environmental Metagenomic DNA sequencing technique was used to shed light on the biodiversity and function of the local nematode community. The technique gives details to find new taxa and genes, construct novel genomes, assess community structure and diversity, and find out which metabolic pathways are encoded in the community (Sharpton, 2014). In this study, environmental metagenomics analysis was performed to determine the diversity of nematodes associated with healthy and unhealthy pineapple plants. Since nematodes are diverse, this study aimed to identify the nematode communities associated with healthy and unhealthy pineapple plants. Thus, it will provide a glimpse of the nematodes that could potentially be pests in pineapple.

Sampling site description
The Pineapple farm sampling site is located at 14°27'31.56588 North and 121°22'6.6 East of Mabitac, Laguna, Philippines, and with an estimated size of 10,000 m 2 where the Red Spanish pineapple variety is grown, a native in the Philippines ( Figure  1-A and B). The area can be characterized by patches of healthy and unhealthy pineapple plants. Soil sampling was done during the pineapple's fruiting stage, around October 2021. The characterization of healthy and unhealthy plants was based on their aboveground symptoms, such as chlorosis and absence or inability to bear fruits.

Soil sample collection
Using the Protocol of Sapkota and Nicolaisen (2015), Soil samples were collected from the rhizospheres of healthy and unhealthy pineapple plants. Three soil samples were collected from each field and were mixed thoroughly. A kilogram of the composite samples was obtained. Subsequently, 1 g of soil for each sampling tube was taken and freeze-dried for 48 hours.

Library construction
DNAs/RNAs were extracted from the samples. Quality control was done, and the samples advanced to library construction. The sequencing library was prepared by arbitrary fragmentation of the DNA or cDNA sample, followed by 5' and 3' adapter ligation. PCR amplification and gel purification followed for the adapter-ligated fragments (Head et al., 2014). To efficiently determine the nematode communities accurately and quantitatively, Next-Generation Sequencing (NGS) was used, where the nucleotide sequences were determined using PCR amplification. DNA sequencing followed, and nematodes were classified based on their DNA barcode sequences (Kenmotsu et al., 2020). Phylogenetic analyses were also done to determine  (Ahn et al., 1996).

Metabarcoding of nematode populations
Using the NGS method in a metagenomic amplicon, samples underwent three stages: the preliminary processing and clustering stage using CD-HIT-OUT (MiSeq FLX), pre-processing and clustering stage using rDNA tools (PacBio), and lastly, the taxonomic assignment and diversity statistics stage (QIIME). In the first stage, sequences were assembled through MiSeqPE using FLASH, producing assembled reads. Filtered reads were clustered at 100% identity using CD-HIT-DUP. Assembled reads were pre-processed and clustered using CID-HIT-OUT to identify chimeras and denoising clusters of size, filtering nonchimeric clusters at a user-specified 97% ID at species level OTU cut-off (Li et al., 2012). Short, ambiguous reads and extra-long tails were removed, including identity chimeras using rDNA tools PacBio (Schloss et al., 2009). The stage generated a distance matrix and clustered using the average neighbor method. Taxonomic assignment and diversity statistics were computed using QIIME using representative sequences from each OTU to assign taxonomy. Phylogenetic trees were constructed from aligned and filtered representative sequences of OTUs. In constructing diversity analysis, Alpha diversity was computed using alpha_diversity.py. Taxonomic compositions were identified using UCLSUT/ RDP (16s or UNITE (ITS) program application. Alphararefaction.py program was used to compute Alpha rarefaction and produced the rarefaction curve graph. PCoA graphs -2D were made using make_2D_plots.py from PCoA. In making the UPGMA tree, upgma_cluster.py was used in the UPGMA tree graph. The heat map was made using the make_otu_heatmap.html.py to produce an OUTheat map. Finally, the publication of quality graphic results was made through statistical analysis and visualization. For a taxonomic assignment, QIIME-UCLUST was used for picking OTUs at 99% similarity since this has been found to be suitable in defining Nematodes OTUs (Porazinska et al., 2012). The RDP 16S rDNA, UNITE-ITS, NCBI-18S, and CO1 were used as references for taxonomic assignments of OTUs. While for diversity analysis and visualizations, QIIME alone was used (Caporaso et al., 2010).

Evolutionary analysis by Maximum Likelihood method
The Maximum Likelihood method and Kimura 2parameter model (Kimura, 1980) were used to create inferences for evolutionary history. The evolutionary tree with the highest log likelihood (-3778.56) was presented, and the percentage of the trees showing the associated taxa clustered together next to the branches was shown. Neighbor-Join and BioNJ algorithms were used and projected by the Maximum Composite Likelihood (MCL) approach for the empirical search of the initial tree(s). A distinct Gamma dispersal was used to determine the evolutionary differences among sites (5 categories (+G, parameter = 0.4215). The analysis involved 37 nucleotide sequences and 538 positions in the final dataset. Evolutionary analyses were then conducted in MEGA 11 (Tamura et al., 2021).

Nematodes associated with both healthy and unhealthy pineapple
Among the nematodes associated with healthy and unhealthy pineapple plants, A. avenae was the most numerous (91.96%). It is a fungivore nematode known to control fungi infestation (Haraguchi and Yoshiga, 2020). They are considered highly active nematodes (Khan et al., 2020), very diverse, and live in all soil habitats. However, they can also be found in leaf covers, root cortex, and crowns, such as in pineapple (Kumari, 2012). They can also be considered free living and found to be associated with plant debris, lichens, cork, and wood tunnels bore by insects (Khan et al., 2020). Its damaging effects on crops are insignificant, but it could be a carrier of potential pathogens such as bacteria and fungi (Kumari, 2012). The result of the study was coherent with the study of (Khan et al., 2020), where A. avenae was the most abundant species (40%). This nematode was also found to be associated with different vegetable crops (Anwar et al., 2013), such as cucumber, tomato, chili, and bell pepper. They stated that A. avenae is an ectoparasite of epidermal root tissue and not considered a dangerous pest of vegetables.

Nematodes associated with unhealthy pineapple plant
In addition, Basiria sp. is one of the plant-parasitic nematodes found in the rhizospheres of unhealthy pineapple plants. It belongs to Family Tylenchidae and is highly diverse in soil habitat. It has been said that the feeding habit of Tylenchidae sp. is indefinite, thus, classifying them as epidermal feeders, mycophagous, or herbivores (Munawar et al., 2021).
While in the study of Kanwar et al. (2021), Basiria sp. served as one of the preys for predatory nematodes such as Mylonchulus dentatus and Aporcelaimllus nivalis and was regarded as plant parasitic. Some of these Brasiria sp. were also found to be associated with pomegranate, fig, grapevine, berry, date palm, lemon, and orange (Azimi, 2019). In Belgium, some species of Basiria were also recorded for the first time on its nematofauna (Bert and Geraert, 2000). This species was also found to be associated with Solanum melongena in Egypt (Ibrahim et al., 2010). In contrast, this species was absent in the rhizospheres of healthy pineapple plants, as results had concluded. Further investigation is recommended to determine the factors resulting in such behavior of the organism. The other species found in the rhizospheres of unhealthy pineapples were classified as unidentified nematodes (EU880054.1). Based on the phylogenetic tree, it has the same lineage as Alaimus sp. (Figure 3) and Rhabdolaimus sp., both free-living nematodes. This species was first reported by Powers et al. (2009) in the Costa Rican Humid Rainforest. They stated that limited nematode diversity was observed in tropical environments rather than temperate ecosystems.

Nematodes found to be associated with healthy pineapple plants
In the case of nematodes found in the rhizospheres of healthy pineapple plants, next to A. avenae is Rotylenchus reniformis (406 and 308/100 g of soil). It is a known-plant parasitic nematode of pineapple (Wang, 2007), particularly an ectoparasitic herbivore (Benzonan et al., 2021;Daramola et al., 2013). Symptoms associated with the infestation done by this nematode would be chlorosis, stunting, plant decline, stunted development, and reduced fruit production and usually associated with a dense population (Soler et. al., 2021). In a study by Sipes and Schmitt (2000), above 1000/g soil of this species would lead to economic yield loss. Evidence of the association with pineapple was reported by Kiriga et al., 2018;Soler et al., 2021. However, it can be noted that the damage could have been more evident since the crop still bore fruits during the time of sampling. Other nematodes found in the rhizospheres of healthy pineapples were Mesocriconema onoense, Aphelenchoides sp., and Ditylenchus gilanicus and considered to be plant parasitic. They were found to be also associated with pineapple (Kiriga et al., 2018; Asian J Agric & Biol. 2023(3). Soler et al., 2021). However, a small percentage was recorded on the soil samples collected. The unidentified nematode (Accession No. EU880147.1) found associated with healthy pineapple plants was first reported again by Powers et al. (2009).
Phylogenetic analysis of the nematodes found to be associated with pineapple Generally, there were only two classes in Phylum Nematoda, Class Chromadorea and Enoplea. There were 16 to 20 orders, and among those, only two orders appeared in the nematode communities associated with pineapple, Rhabditida, and Enoplida. However, the family Aphelenchoididae from the Order Rhabditida is distant from the lineage because of its polyphyletic nature. Order Rhabditida comprises many bacterivores, fungivores, Phytofeeders, and parasites of vertebrate and invertebrate animals. It thrives in soil or freshwater niches (Abolafia and Peña-Santiago, 2007). As mentioned, (Ruiz-Cuenca and Abolafia, 2022), there are around 450 genera under this Order. However, numerous phylogenetic studies have been published and challenged the classification (Ahmed and Holovachov, 2021) since nematodes are very diverse and evolving. In the family Anguinidae, only the species Ditylenchus gilanicus was found. It is a new species discovered in Iran and belongs to the D. triformis group (Yaghoubi et al., 2018). This species is characterized by a rounded tail tip and lateral field of six lines and is considered a fungivore (Qiao et al., 2016). In Apelenchidae family, only the species Apelenchus avenae was found to be associated with pineapple. It is also a fungivore characterized by a white body, tapered but rounded on both ends, with a short style. It has a short stylet, and metacorpus fills the whole-body diameter, which is the distinguishing characteristic of this species (Kumari, 2012). As discussed by Karim et al., 2009, this nematode evolved to have a set of genes encoding a range of enzymes that could degrade cell walls. These findings suggest that it could also be a plant parasite. In the family of Cephalobidae, two species were found, Acrobeloides varius and the unidentified nematode (132). The species are known to share other characteristics as that of other Acrobeloides sp., including an elongated swelling corpus and lateral incisures ranging from 1-5µ that extend up to the pointed tail (Kim et al., 2017). This species is also known to be a bacterial feeder (Trap et al., 2021). Thus, the unidentified nematode, which is 98% identical to A. varius, both found in the rhizospheres of the soil samples, were categorically considered a bacterial feeder. Another family in Order Rhabditida is Criconematidae. They are also known as ring nematodes and are considered plant parasitic. They are also considered monophyletic because of their distinguishing characteristics, such as the moprodelphic ovary without a post-vulva sac (Powers et al., 2017). Mesocriconema onoense was one of the plant-parasitic nematodes found in the soil samples. It is one of the 90 species in the genus Mesocriconema and is classified as an ectoparasitic root nematode (Nguyen et al., 2021). The same nematode species was also observed in Vietnam (Nguyen et al., 2021) and reported by (Powers et al., 2017). Hoplolaimidae is the family to which Rotylenchulus reniformis belongs. The species is also known as reniform nematodes and is semiendoparasitic. It is also one of the ten species under the genus Rotylenchulus. The species is 100% identical to that found in the farm soil in Alabama (Nyaku et al., 2016). Basiria sp., on the other hand, is generally considered plant parasitic. It belongs to the family Tylenchidae, one of the most diverse and numerous nematodes habiting in soils. It includes 42 genera, including Basiria sp. Each species under this genus has different characteristics and is monophyletic or belongs to a single common ancestor (Eisvand et al., 2019). It was described by Bert et al. (2003) as "small to medium size nematodes, with an oral opening surrounded by six papillae with a second circle of four papillae on the edges of the anterior surface." Mostly, the tails are elongated with a rounded end, seldom spicate. Another nematode under the Order Rhabditida is Panagrolaimoidea sp., which is 100% identical to that found in the dissected body of Coatitermes clevelandi, a termite (Kanzaki et al., 2012), thus an insect-associated nematode. This genus belongs to the Family Cephalobina. Andrassy (2005) reported that this predominantly includes bacterivores that show greater phylogenetic kinships to Tylenchids, which is also the same Order as the latter (Khan et al., 2012). Rhabditid nematodes can be found abundantly in different soil types and sediments, whether on land or freshwater. They can be free-living bacterivores of saprovores or entomopathogenic parasites. Generally, nematodes belonging to the Order Rhabditida can be considered as a challenging nematode group where diversity is highly far from being prominent (Abolafia and Peña-Santiago, 2007). Enoplida is one of the two nematode orders associated with pineapple. Only two of the 21 families under this order were associated with the crop: Family Alaimidae and Rhabdolaimidae. Most of the nematodes in this order can be found in marine or moist land environments. They are commonly classified as predators (Andrassy, 2005). Specifically, two species of nematodes (Alaimus sp. And Rhabdolaimus aquaticus) and three unidentified nematodes were found in the rhizospheres of pineapples. Alaimus sp. is a bacterivore nematode often associated with mosses (Andrassy, 2005). Rhabdolaimus aquaticus is also a bacterivore and generally thrives in riverbanks or moist locations (Botha A and Heyns J, 1993). The three other unidentified nematodes were with the same lineage (94-100% similarity) as those found in the humid lowland of Costa Rica and were most likely a bacterivore. Two nematode species of the Order Rhabditida belonging to the family Aphelenchoididae were also found in the pineapple rhizospheres. These were separated from Rhabditida since Apelenchoididae is polyphyletic (Esmaeili et al., 2017), coming from different ancestral lineages. Aphelenchoinidae sp. is often associated with monocots, gymnosperms, and ferns (Aliramaji et al., 2018). Aphelenchoides sp., on the other hand, is composed of fungivores and plant parasites. Sometimes, the parasitic species are referred to as foliar nematodes since they feed primarily on leaves and buds (Duncan and Moens, 2013). The species found in the soil sample was 46% like that found in Belgian soil, which was found by Sánchez-Monge et al. (2017), a plant parasite. However, according to by Sánchez-Monge et al. (2017), there was a significant number of undescribed species in the Family Aphelenchoididae and increasing the number of taxonomic studies and evolution will shed light on the biodiversity of this family.

Conclusions
Metabarcoding of nematode communities associated with pineapple provided insights on its diversity. Fungivores, bacterivores, predators, and plantparasitic and insect-associated nematodes were present. The presence of plant-parasitic nematodes in the rhizospheres of the crop did not yet pose a significant threat since their density is still below the level of infestation. Nevertheless, it showed a glimpse of the plant-parasitic nematode community, which could be detrimental to pineapples.