Diversity and Characterization of Plant Parasitic Nematodes Associated with Cereals in Haiti

Thesis

Diversity and characterization of plant parasitic nematodes associated with cereals in Haiti

Lesly Joseph Student number: 01700927

Promoter: Prof. Dr. Wim Bert

A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of International Master of Science in Agro- and Environmental Nematology

Academic year: 2018 -2019

Diversity and characterization of plant parasitic nematodes associated with cereals in Haiti

Lesly Joseph Nematology Research Unit, Department of Biology, Ghent University Ledeganckstraat 35, Ghent 9000, Belgium

Abstract The diversity and the prevalence of plant parasitic nematodes associated with the cereals in Haiti have been assessed from samples collected in 35 agricultural fields: rice (18 samples), maize (11 samples) and sorghum (6 samples). Thirty (30) nematode species belonging to 12 genera have been identified. From the nematode species recovered, 20 nematode species from 12 genera were found associated with rice, 15 nematode species from 9 genera with maize and 9 nematodes species from 7 genera with sorghum. Five new species have been identified, from the following genera Helicotylenchus (2), Pralylenchus (1), Xiphinema (1) and Ditylenchus (1). From the nematode species recovered within the rice fields, Meloidogyne graminicola, Pratylenchus zeae, Helicotylenchus retusus were the most prevalent and abundant. Within the maize fields, Pratylenchus parazeae, Helicotylenchus erithrinae and Rotylenchulus reniformis were the most prevalent and abundant. In the sorghum fields, Pratylenchus parazeae, Helicotylenchus erithrinae and the criconematids were the most abundant nematode species but at low prevalence. This study confirmed the evidence that the prevalence and the density of plant parasitic nematodes are influenced by the crops. The sorghum cultivars in the investigated fields are poor host for the most damageable plant parasitic nematodes. Nine nematodes species (Tylenchorhynchus agri, Rotylenchulus reniformis, Pratylenchus parazeae, Pratylenchus zeae, Meloidogyne graminicola, Helicotylenchus erythrinae, Helicotylenchus retusus, Quinisulcius acutus, Mesocriconema sphaerocephalum) have been characterized morphologically and molecularly. This study provided the first molecular characterization and the first sequences for Helicotylenchus erythrinae, Helicotylenchus retusus and Quinisulcius acutus based on the 18S rDNA, D2-D3 and ITS1-5.8-ITS2 regions. The first sequences of the 18S rDNA gene for Tylenchorhynchus agri and Xiphinema basiri are provided from this research. This study presented an overview of the phylogeny of the sub-family of Telotylenchinae and suggested the combination of the genera Paratrophorus, Bitylenchus, Neodolichorhynchus, Sauertylenchus, Histotylenchus, Telotylenchus, Quinisulcius with Tylenchorhynchus to form a super genus of Tylenchorhynchus sensu lato. Key words: Plant parasitic nematode, cereals, phylogeny, species, Haiti, maize, rice, sorghum

Introduction Plant-parasitic nematodes are omnipresent in all agricultural areas in the world. Globally, they are responsible of 10% of the total losses in the agricultural production chain (Savary et al., 2012; De Waele & Elsen, 2007). The monoculture and the conventional agriculture increase the density and the prevalence of plant of plant-parasitic nematodes (Coyne and al, 1999; Coyne and al, 2001). The biggest density of plant of plant parasitic nematodes has been observed in the ecosystem of the most cultivated crops in the

2 world (cereals, potatoes, soybean) (Sikora and al, 2018), which consequently induced more damages, more losses. According to FAO (2012), 30% of the total cereals produced in the world are lost. Taking into consideration the estimation of De Waele & Elsen (2007), plant parasitic nematodes are responsible of 3% of the total cereal lost worldwide. The cereals are staple foods for more than half of the human population, who are dependent at daily basis for their energy uptake (FAO, 2014). With the increasing human population, there is an increasing gap between the total cereals produced and the needs for those crops, which tend to affect the food security of the population of many third countries which are highly dependent on.

In the Caribbean region, Haiti is among the countries with the highest rate of consumption of cereals, particularly rice (Sanou et al., 2013). Every year, on average an Haitian eat 40 kg of rice and on average 20 kg of maize and sorghum (Baro, 2002; Locher, 2001) . However, even though the cereals are crucial for the alimentation of the Haitian population, the country suffer a huge deficiency in the tonnage consumed compare to its production. In Haiti, more than 400 000 tons of rice are consumed yearly, while the annual production is around 172 000 tones, which represent 43% of the total needs and the rest are imported. The production of maize in Haiti is around 251 000 tons per year, which represent 90% of the total maize used for human consumption in the country (Dumazert & Ernest, 2017). Perhaps, the maize used as feeds are mainly imported, that constitute a barrier for the improvement of the livestock production in the country. Haiti is self-sufficient for sorghum supply for human consumption (ACF, 2017). However, with the increasing use of sorghum in beer production in Haiti, efficiency in the production should improve to prevent shortage. The agricultural system of Haiti is characterized by a low productivity and a low efficiency in the natural resources used in the production chain. Too many production factors are limited or missed in the production chain. The structural constraints intrinsic to this agricultural system, make plant parasitic nematodes not really regarded as a major problem that weaken the sustainability of this system of production. Therefore, plant parasitic nematodes don’t receive the attention they deserved in Haiti considering their known impacts in the agricultural sector worldwide. Furthermore, the lack of active nematologists and the low investment globally in agricultural researches from the government are also responsible of the sparsity of researches in the nematology field in Haiti. Consequently, most of the nematodes (Plant parasitic and free living) inhabitant the Haitian territory remain unknown to the scientific community. The first published report of plant of plant parasitic nematodes in Haiti was submitted by Crill (1973), exploring the diversity of plant parasitic nematodes associated with tomatoes in Haiti. Later, CIRAD (2005, 2012) presented Pratylenchus and Meloidogyne amongst the pests of yam and plantains in Haiti. Beside these two reports, no publication has been recovered about plant parasitic nematodes in Haiti. To our knowledge, none plant parasitic nematode from Haiti has already morphologically and molecularly characterized or link to molecular barcoding. Knowing that nematodes are omnipresent and caused important losses in the tropical regions where Haiti is, effective taxonomy strategy to identify the plant parasitic nematodes associated with the cash crops of Haiti at species level are an emergency now. The assessment of the diversity and the damage thresholds of the most abundant species will probably help to develop effective management strategy. The integrative taxonomy, combining traditional taxonomy and molecular barcode, is so far the best method to identify plant parasitic nematodes, as it combines the morphological and the genetic variations to determine the taxonomical status of a specimen or a population (Palomares-Rius et al., 2017). The molecular barcode in nematology is relevant, as it facilitates the identification of nematodes by non-experts in this field and globalized the research on the phylogeny of this order. The molecular barcode method could facilitate taxonomical work in the south countries like

Haiti, where expertise on nematode taxonomy are missing for the identification of the nematode morphologically. The general objectives of this study were to evaluate the density and the prevalence of the plant parasitic nematodes associated with rice, maize and sorghum and Provide morphological and molecular characterizations of the most important species recovered, including informative molecular barcodes for the partial 18S SSU rDNA, D2-D3 28S LSU rDNA and the ITS genes.

Materials and methods Samplings and extraction During November 2018, samples of soils and roots where collected in 35 agricultural fields from maize (11 samples), rice (18 samples) and sorghum (6 samples) in Haiti (Fig 1). For each field, the geographical position coordinates were taken using a GPS Garmin. The samples were collected by digging out one plant and collect the soils attached and surrounding the roots system into a plastic bag. Soil samples were processed from a representative 200 ml using a modified Baermann funnel method. The nematodes recovered were fixed in DESS solution (DMSO-EDTA salt-saturated solution) in Haiti. The remaining part of each sample was brought to Belgium. In February 2019, 200 ml of soil from these samples were processed to extract the nematodes by using a modified Baermann funnel method for 72 hours and the nematodes recovered where fixed in Formalin. The Baunacke method was used for the extraction of the cyst nematodes in Belgium.

Fig 1: Map presenting the locations of the samples collected in Haiti

Density and prevalence value

The density of the different genera of PPN in each sample was calculated by pouring all the nematodes extracted into a counting dish of 100 squares. The plant parasitic nematodes within the two principal diagonals of the counting dish (20 out of 100 squares) were identified at genus level and counted for the estimation of the density of each genus per sample, using a stereomicroscope. The prevalence value, showing the frequency of occurrence of every genus has been calculated using the formula below. The results were interpreted based on the thresholds fixed by FORTUNER & MERNY (1973) , where a genus was classified as frequent for an occurrence value at least 30% and rare for an occurrence value less than 30%. The densities of the PPN were classified as weak (5PPN/100ml), as average (20 PPN/100ml), as high (100 PPN/100ml) and as very high (500 PPN/100ml). Mann-Whitney Rank Sum Test (Wallace, 2004) was used to detect the significance of the difference observed between the overall prevalence and density of plant parasitic nematodes within the samples of the three (3) crops. 푇표푡푎푙 푛푢푚푏푒푟 표푓 푓푖푒푙푑푠 푝표푠푖푡푖푣푒 푓표푟 푎 푔푒푛푢푠 X 100 푃푟푒푣푎푙푒푛푐푒 = 푇표푡푎푙 푛푢푚푏푒푟 표푓 푓푖푒푙푑푠 푠푎푚푝푙푒푑

Morphological characterization The nematodes fixed in formalin and DESS were transferred to anhydrous glycerin and mounted in glass slides with cover glass supported by a wax ring. The nematodes were examined, measured, photographed and drawn by using an Olympus BX50 DIC Microscope (Olympus Optical), equipped with an Olympus C5060Wz camera and a drawing tube. Molecular analyses The DESS-preserved nematodes, rinsed in distilled water for about 60 minutes, were individually picked and mounted on temporary glass slides to record all necessary morphological and morphometric data by taking pictures and measurements as described above. DNA extraction was done by cutting an individual specimen and transferring it to a PCR tube with 20 μl of worm lysis buffer (50 mM KCl, 10 mM Tris at pH 8.3, 2.5 mM MgCl2, 0.45% NP 40 (Tergitol Sigma), 0.45% Tween-20) followed by incubation at −20°C (10 min), adding 1 μl proteinase K (1.2 mg ml −1), incubation at 65°C (1 h) and 95°C (10 min) and ﬁnally centrifuging the mixture at 14000 g for 1 min. To amplify the target genes, 2 µl of gDNA suspension was used as template in a 23 µl PCR reaction mix (TopTaq Qiagen, Germany). The primers set used in this study to amplify the targeted genes (18S rRNA, 28S rRNA, ITS1-5.8-ITS2 rRNA, COI) are presented in table 1. Amplification cycles were conducted in the Thermocycler Eppendorf programmed with the following thermal profile: starting with 94°C for 4 min; followed by 40 cycles at 94°C for 30 sec; annealing temperatures starting at 58°C (18S and D2D3), 60°C (ITS) and 45°C (COI) for 30 s; 72°C for 1 min and ﬁnished at 10°C. Aliquots of 5 µl of the PCR products were sized with low DNA mass ladder and separated by electrophoresis in 1% agarose gel stained with GelRed and observed using a UV Transilluminator

Table 1: Primers sets used in this research

Genes Forward primers Reverse primers References 18S SSU18A SSU26R Mayer et al 2007 rRNA 5'-AAAGATTAAGCCATGCATG-3’ 5'-CATTCTTGGCAAATGCTTTCG -3' 28S D2A D3B (De Ley et al., 1999) rRNA 5’-ACAAGTACCGTGAGGGAAAGTTG-3’ 5’-TCGGAAGGAACCAGCTACTA-3’ MalF 1006R (Qing et al., 2017) 5’-GGATAGAGCCRACGTATCTG-3’ 5’-GTTCGATTAGTCTTTCGCCCCT-3’ ITS1-5.8- Vrain2F Vrain2R (Elbadri et al., ITS2 5’-CTTTGTACACACCGCCCGTCGCT-3’ 5’-TTTCACTCGCCGTTACTAAGGGAATC-3’ 2002; Vrain, 1993) rRNA TW81 AB28 (Subbotin et al., 5’-GTTTCCGTAGGTGAACCTGC-3’) 5’-ATATGCTTAAGTTCAGCGGGT-3’ 2017) COI JB3_prat JB4_prat (Janssen, 2017) 5’-TTTTTTGGGCATCCTGAAGTCTAT-3’ 5’-CCTATTCTTAAAACATAATGAAAATG-3’ JB3 JB4 (Palomares-Rius et 5’-TTTTTTGGGCATCCTGAGGTTTAT-3’ 5’-TAAAGAAAGAACATAATGAAAATG-3’ al., 2017)

Sequence alignment and phylogenetic analysis The obtained sequences for the ampliﬁed genes were assembled using assemble de novo in Geneious (Kearse et al., 2012). The sequences of each genus were aligned with subsequent sequences of the same genus available in GenBank by using Muscle in MEGA7 (Edgar, 2004). Muscle was run with the default parameters and the analysis were in full length alignment. Sequence alignment were analyzed with Bayesian inference (BI). For each gene, the Bayesian Inference analysis under the GTR + G + I model was initiated with a random starting tree and two separate runs of four chains for two million generations. The Markov chain Monte Carlo (MCMC) were sampled at interval of 100 generations. A 50% majority consensus tree was generated after discarding a burn-in samples of 15%. The posterior probability is given in decimal for each node. Trees were visualised and rooted using FIGTREE v1.4.

Results

Prevalence and density of plant-parasitic nematodes

Within the investigated fields of cereals (rice, maize, sorghum) in Haiti, a total of 30 species of plant parasitic nematodes belonging to 13 genera have been identified (Table. 2). Rice presented the highest nematodes diversity with 20 species belonging to 12 genera, following by Maize with 15 nematodes species belonging to 9 genera, and sorghum with 9 nematodes species belonging to 7 genera (Table. 2). Around 90% of the nematode species associated with maize and sorghum are parasites of rice. Six new species were found, from the following genera Helicotylenchus (2), Pralylenchus (1), TYlenchorhynchus (1), and Xiphinema (1), Ditylenchus (1). The genera Meloidogyne, Helicotylenchus and Pratylenchus were the most prevalent and the most abundant plant parasitic nematodes associated with the three cereals crops (rice, maize, sorghum) (Fig. 2 & Fig. 3). The relative abundance of the genera differed between the crops. Within the investigated rice fields, the genus Meloidogyne presented the highest density (174 ± 111 x 100 푚푙−1) following by Helicotylenchus (154 ± 132 x 100 푚푙−1). Over the maize samples, Pratylenchus (87±42,3) and Helicotylenchus (67,1 ± 30,4 x 100 푚푙−1) were the most abundant genera. The Rotylenchulus species were maximally prevalent in the maize fields but at low density. Amongst the sorghum nematodes recovered,

6 the Criconema (100 ± 20 x 100 푚푙−1), Pratylenchus (70 ± 36 x 100 푚푙−1) and Helicotylenchus (65 ± 21 x 100 푚푙−1) were the most abundant. The genera (Tylenchorhynchus, Quinisulcius, Mesocriconrma, Xiphinema, Geocenamus) were less prevalent and more abundant in the rice fields compared to maize and sorghum. The genus Helicotylenchus was, with height (8) species identified, the most diverse and the most widespread genus over the investigated fields in this study. The common rice nematodes of the Hirschmanniella genus were absent amongst the nematodes recovered from the rice fields in Haiti. Five (5) Globodera cyst and seven (7) Heterodera cyst have been found respectively from the rice and the maize fields. Unfortunately, no juvenile had been recovered for further analysis. The difference between the three crops were not significant for the overall prevalence of plant parasitic nematodes associated with them separately. Although, the densities of the nematode species associated with rice were significantly higher than maize (rice & maize, p= 0,012) and sorghum (rice & sorghum, p= 0,032).

120

100

60 Rice Maize 40 Sorghum

Prevalence % Prevalence

20 Rice - maize: p= 0,43

Rice-Sorghum : p= 0,93 0 Maize-sorghum: p= 0,76

Xiphinema Criconema Ditylenchus Quinisulcius Meloidogyne Rotylenchus Pratylenchus Geocenamus Rotylenchulus

Helicotylenchus Mesocriconema

Tylenchorhynchus

Fig 2: Mean of prevalence of plant parasitic nematode genera associated for the three cereal crops investigated. The mean is the average of prevalence of each genus in respectively 16 rice fields, 9 maize fields and 5 sorghum fields. p-value of the Mann-Whitney rank Sum Test testing the significance of the difference in the prevalence of PPN within the samples of the three crops. The symbol is the average? Significance test nt clear, above you describe significant diffrences

Table 2: List of nematodes species recovered in this study

Rice -maize : p= 0,012

Rice-Sorghum : p= 0,032

Maize-sorghum : p= 0,88

Fig 3: Mean of the density of plant parasitic nematode genera associated with the three cereal crops investigated. The mean is the average density of each genus in each field separately of the 16 rice fields, 9 maize fields and 5

sorghum fields. p-value of the Mann-Whitney rank Sum Test testing the significance of the difference in the density of PPN within the samples of the three crops. mention direction of significant differences, for example rice significant higher density than ..

Morphological and molecular characterizations Within the samples collected in the three cereals crops in Haiti, using traditional morphological taxonomic characters integrated with molecular criteria, we distinguished thirty (30) valid species (Table 2). From the identified nematode species, molecular characterization based on the D2-D3 of the 28S rRNA, ITS, partial 18S rRNA and COI genes combined with morphometric and morphological characterization are given for nine (9) species, containing: Tylenchorhynchus agri, Rotylenchulus reniformis, Pratylenchus parazeae, Pratylenchus zeae, Meloidogyne graminicola, Helicotylenchus erythrinae, Helicotylenchus retusus, Quinisulcius acutus, Mesocriconema sphaerocephalum. The specimens of the hypothetical new or undescribed species, including the genera Ditylenchus, Geocenamus, Rotylenchus, were not identified at species level. These populations did not contain sufﬁcient number of specimens for their morphological identiﬁcation and determination of their taxonomic status.

1- Tylenchorhynchus agri Ferris, 1963 Morphological characterization Specimens belonging to Tylenchorhynchus agri Ferris, 1963 had been recovered from four (4) rice fields in Haiti. Female body slightly arcuate ventrally. Body cuticle with distinct annulation, measuring anteriorly 1,33 µm, in mid body and on tail 2,42 µm. Lateral fields about 25% of the body width marked with four incisures, the outer incisures distinctly crenated. Lip region slightly off-set bearing four (4) annules. First neck annule posterior to head is slightly reduced. Stylet with basal knobs concaved anteriorly set-off from the shaft. Median bulb ovoid, at 9% of the total body length from the anterior end. Basal bulb elongated, pyriform, not overlapping the intestine. Secretory-excretory pore and vulva respectively at 14,19% and 54,18% of the body from the anterior end. Spermatheca relatively small, ovoid, either empty or filled with sperm. Post anal sac occupied more than 50% of the total length. Tail sub-cylindrical with 18-20 annuli, tail terminus sub-hemispherical, smooth. Phasmid posterior to level of anus, 37,5 - 39% of tail. The males were absent in the samples.

Molecular characterization Three 854-bp of the partial 18S rRNA products were sequenced, based on DNA template of three single females recovered from rice fields in Haiti. The Intraspecific variations of the 18S sequences obtained for this population of T. agri were 0,2-0.5% (2 - 4 bp). A Blastn search of the generated sequences revealed 99,0% similarity with the deposited sequences of T. zeae into GeneBank (MG961204, KJ461619). The Baysian inference phylogenetic tree (Fig. 3) based on the 18S rRNA gene; from a multiple alignment of 821 characters, showed T. agri in a maximally supported monophyletic group. This study provided the first sequences of T. agri for 18S rRNA gene. Four 753-bp of the 28S rRNA products were sequenced, based on DNA template of four single females recovered from rice fields in Haiti. The Intraspecific variations of the 28S sequences obtained for this population of T. agri were 0,0-0.5% (0 - 3 bp). A Blastn search of the generated sequences revealed 99,8- 100% similarity with the deposited sequences of T. agri into GenBank (MG560824, KX622690 and MG491667). The inter-specific variation of the D2-D3 of the 28S rRNA region between the obtained sequences and the deposited sequences of T. agri into GeneBank were 0 - 4,6% (0-27 bp). The Baysian inference phylogenetic tree (Fig. 3) based on the 28S rRNA gene; from a multiple alignment of 719 characters, showed T. agri formed a maximally supported monophyletic group with the other T. agri sequences and two uncharacterized Tylenchorhynchus sequences (KJ461559, KJ461559). The T. agri clade was sister taxon with a maximally supported monophyletic clade of T. leviterminalis. Three 863-bp sequences of the rDNA ITS gene were obtained, based on DNA template from three single females recovered from rice fields in Haiti. The Intraspecific variations of the rDNA ITS sequences of this population of T. agri were 0,3-1.0% (3 - 9 bp). A Blastn search of the generated sequences revealed 98,94- 99,16% similarity with three deposited sequences of T. annulatus (EF030983, MG430283, MH142616) and 98,64 – 99,35% similarity with three uncharacterized Tylenchorhynchus sequences (KJ461591, KJ461592, KJ461593). The inter-specific variations of the sequences generated in this study were 0,3-1,3 (3-11 bp) and 0,2 -1,4% (2 – 12 bp) respectively for the T. annulatus and the Tylenchorhynchus sp. sequences mentioned above. The Baysian inference phylogenetic tree based on rDNA ITS gene (Fig. 3 -C); from a multiple alignment of 834 total characters, showed the sequences of T. agri generated in this study formed a maximally supported monophyletic clade wit the T. annulatus and the Tylenchorhynchus sp. sequences previously mentioned. And, according to molecular data, conspecific with Tylenchorhynchus agri study.

Remarks The morphology and the morphometrics data of the Tylenchorhynchus agri population recovered in this study are in agreement with the original description (Ferris, 1963). However, the Haitian population of T. agri have a larger body width (23,74 µm ± 3 vs 21 µm) and a longer tail (42 – 53 µm vs 32-39 µm) compared to the original description. In comparison with the specimens studied by Ciobanul (2004) in Romania, the body length of the Haitian population is remarkably smaller (538-695µm vs 726–730µm). Tylenchorhynchus agri, in respect to the original description, appears to be very plastic morphometrically taking into consideration the huge morphometric variability amongst several populations reported from many places in the world (Anderson & Potter, 1991; Bernard , 1980; Knobloch & Bird , 1981). Morphologically T. agri is faintly distinguishable from T. ewingi (Ferris VR, 1963; Geraert, 2011), both have a post anal intestine sac but the tail shape of the female (subcylindrical vs conoid) and the number of tail annuli ( 21 vs 15) differentiated the two species. Unfortunately, molecular data are not available for T. ewingi to analyse its phylogenetic relationships to T. agri. Morphologically, T. annulatus shows huge similarity to T. agri but they are different for the position of the post anal intestinal sac (5-10% vs 30-50%), spermatheca (not discernible vs visible and filled) and the occurrence of male (absent, present). Based on the phylogenetic analysis of the partial 18S rRNA, D2-D3 of the 28S rRNA and the ITS genes, where T. annulatus formed a maximally supported clade in the Baysian inference tree for these genes separately, we assume that the specimens of these sequences (EF030983, MG430283) highlighted in yellow in the ITS tree ( Fig. 3 – C) were identified mistakenly respectively by Chen and al (2006) and Solangi and al (2017) as T. annulatus, therefore should be consider as T. agri . Handoo et al ( 2014) have already suggested the consideration of T. annulatus (EF030983) as T. agri. The specimens identified as Tylenchorhynchus sp (KJ461591, KJ461592, KJ461593) by Handoo et al (2014), highlighted in green in the Fig. 3 – C, belong to T. agri based on molecular and morphological data recovered in this study. These specimens were identified properly as T. agri in the publication of Handoo et al ( 2014) while they deliberately identified them as Tylenchorhynchus sp. In GenBank.

Table 3. Measurements of females of Tylenchorhynchus agri on rice in Haiti and other populations. All measurements are in µm and in form: mean ± SD (range), except for the ratio

A: 18S

A : 28S

C: ITS

Fig. 4. Phylogenetic trees of Dolichodorids species based on 18S (A), D2D3 28S rDNA data (B) and ITS1- 5.8-ITS2 rDNA data (B) obtained from Bayesian inference (BI) performed with MrBayes v3.1.2 using a GTR + I + G model. Branch support is indicated with PP: high (≥95% PP)/ medium (>95% to ≥ 50% PP)/ low (<50% PP).

Fig.5. Tylenchorhynchus agri. LM of female. A: Posterior region; B: Anterior region; C: Lateral lines; D: Entire female body; E: Vulva region. (Calibration: A, B, C= 100 µm; D= 20 µm; E= 20 µm)

Rotylenchulus reniformis Linford & Oliveira, 1940 Morphological characterizations Description of immature females Lateral field less than 25% of the body width, not areolated. Body in open C shape. Lip region conoid, not offset from body, conspicuous with 5 annules. Stylet with stylet knobs rounded, slope backward. Excretory pore at 19% of the body length from anterior end. Metacorpus elongate, oval. Pharyngeal glands overlap intestine lateral-ventrally. Vulva post-median from the anterior end. Female reproductive system amphidelphic with two flexures. Tail terminus rounded with hyaline part. Description of males Body generally curled. Lateral field less than 25% of body width, not areolated. Lip region conoid, slightly sclerotized, not offset. Stylet weak with stylet knobs rounded. Metarcorpus oval at 12,7 % of the body length from the anterior end. Excretory pore at 17,8 % of the body length from the anterior end. Pharynx regions faintly distinct. Spicule thin, slightly curved. Tail elongated with hyaline part. Mature females not recovered in this study. Molecular characterizations Four 730-bp of the partial 18S rRNA products were sequenced, based on DNA template of four single females recovered from two maize fields in Haiti. The Intraspecific variations of the 18S sequences obtained for this population of R. reniformis were 0,3-1.1% (2 - 7 bp). A Blastn search of the generated sequences revealed 98,8-99,0% similarity with the deposited sequences of Rotylenchulus reniformis into GeneBank (KP054067, KP054062). The inter-specific variation of the Partial 18S rRNA gene between the obtained sequences and the deposited sequences of R. reniformis into GeneBank were 0,7 - 8,6% (5-62 bp). The Baysian inference phylogenetic tree (Fig. 7-A) based on the 18S rRNA gene; from a multiple alignment of 701 characters, placed the R. reniformis from Haiti amongst the other sequences of R. reniformis deposited into GeneBank in two different clades maximally supported. Two 596-bp of the 28S rRNA products were sequenced, based on DNA template of two single females. The Intraspecific variations of the 28S sequences obtained for this population of R. reniformis were 0.8% (5 bp). A Blastn search of the generated sequences revealed 98,83-99,4% similarity with the deposited sequences of R. reniformis into GenBank (MH136563 and KP054120). The inter-specific variation of the D2-D3 of the 28S rRNA region between the obtained sequences and the deposited sequences of R. reniformis into GeneBank were 0,8 - 17% (5-101 bp). The Baysian inference phylogenetic tree (Fig. 7-B) based on the 28S rRNA gene; from a multiple alignment of 590 characters, placed the R. reniformis from Haiti amongst the other sequences of R. reniformis deposited into GeneBank in a maximally supported clade. Remarks orphologically and morphometrically the populations of Rotylenchulus reniformis from maize fields in Haiti are congruent with the description of the type species (Linford & Oliveira, 1940) and the neotype (Dasgupta and al, 1968). Morphologically, the Rotylenchulus reniformis populations in this study fall within the range of variation of other populations reported in other countries, with a tendency toward a smaller sizes compare with the US populations (Agudelo et al., 2005) and aligned with the morphometrics data of the African population (Germani G., 1978).

A: 18S

B: 28S

Fig. 6. Phylogenetic trees of Rotylenchulus reniformis based on (A) Partial SSU18S and (B) D2D3 LSU 28S rDNA obtained from Bayesian inference (BI) performed with MrBayes v3.1.2 using a GTR + I + G model. Branch support is indicated with PP: high (≥0.95 PP)/ medium (>0.95to ≥ 50% PP)/ low (<50% PP).

Fig.7. Rotylenchulus reniformis. LM of male and female. A: Entire immature female; B: Posterior region immature female; C: Entire male; D: Anterior region male; E: Posterior region male. (Calibration: A, B, E= 40 µm; c= 10 µm; E= 100 µm).

Pratylenchus parazeae Wang et al., 2015

Morphological characterization Female body shape straight to some extent curved ventrally. Lateral field occupies 20% of corresponding body diameter with four incisures, areolated in outer bands. Labial framework heavily sclerotized bearing 3- 4 annuli. Stylet robust, very long shaft, 47–52% of entire stylet long. Dorsal pharyngeal gland opening at 2.66 ± 0.49 μm posterior to the stylet base. Median bulb well developed, oval. Excretory pore at cardia level, 48.96 ± 3.31 μm from the anterior end. Pharyngeal gland lobe ventrally overlapping intestine, with three gland nuclei: two latero-ventral and a ventral one. Vulva post-median, vulva lips slightly protruding. Reproductive tract monodelphic, germinative zone outstretched. Spermatheca small, oval and empty. Postvulval uterine sac 20-24 % of the vulva-anus distance. Phasmids pore like, 39,5 % of the tail length from the anus. Tail subcylindrical with rounded to bluntly pointed terminus, tapering toward tip, bearing 23–32 annuli.

Molecular characterization Thirteen 930-bp of the partial 18S rRNA products were sequenced, based on DNA template of Thirteen single females recovered from seven maize fields in Haiti. The Intraspecific variations of the 18S sequences obtained for this population of Pratylenchus parazeae were 0,0-2% (0 - 119 bp). A Blastn search of the generated sequences revealed 98,72-99,19% similarity with the deposited sequences of Pratylenchus parazeae into GeneBank (KP903437, KP903432). The inter-specific variation of the Partial 18S rRNA gene between the obtained sequences and the deposited sequences of P. parazeae into GeneBank were 0,01 - 2,7% (9-27 bp). The inter-specific variations of the Partial 18S rRNA gene between the obtained sequences of P. parazeae and the sequences of P. zeae generated in this study were 4 - 8% (37-74 bp). The Baysian inference phylogenetic tree (Fig. 7-A) based on the 18S rRNA gene; from a multiple alignment of 902 characters, placed the P. parazeae from Haiti amongst the other sequences of P. parazeae (including the sequence of the type specimen) deposited into GeneBank in a maximally supported clade. The monophyletic clade of P. parazeae was sister taxon with Pratylenchus zeae with the maximum support. From the collected population of Pratylenchus parazeae of this study, seven 605-bp of the 28S rRNA products were sequenced, based on DNA template of seven single females from 4 different fields. The Intraspecific variations of the 28S sequences obtained for this population of P. parazeae were 0 - 1.3% (0 – 8 bp). A Blastn search of the generated sequences revealed 98,44-100,0% similarity with the deposited sequences of P. parazeae into GenBank (KP903440, KF765435). The inter-specific variation of the D2-D3 of the 28S rRNA region between the obtained sequences and the deposited sequences of P. parazeae into GeneBank were 0,0 – 1,4% (0-8 bp). The Baysian inference phylogenetic tree (Fig. 7-B) based on the 28S rRNA gene; from a multiple alignment of 600 characters, placed the P. parazeae from Haiti amongst the other sequences of P. parazeae deposited into GeneBank in a maximally supported clade.

Table 5. Measurements of females of Pratylenchus parazeae on maize in Haiti and other populations. All measurements are in µm (except for ratio) and in form: mean ± SD (range)

Character Haiti Baise city, Guangxi, China Laibin city, Guangxi, China Hechi city, Guangxi, China Female Holotype paratype females females females female n 10 20 20 20 Lb 453.5 ± 53,08(363-509) 562.9 587.5±37.8 (527.8–682.6) 628.1±43.0 (532.2– 649.1±26.1 (600.0–697.3) 704.7) a 21.47 ± 1.88(18,8-24,2) 23.1 24.7±1.8 (21.6–28.2) 29.7±1.7 (25.7–33.8) 26.9±1.8 (24.1–30.3) b 6.34 ± 0.93(5,18-8) 6.7 6.4±0.5 (5.6–7.7) 7.0±0.5 (6.2–7.9) 7.0±0.4 (6.1–7.7) b’ 4.32 ± 0.83(3,4-5,9) 4.7 4.5±0.3 (4.1–5.2) 4.5±0.3 (3.7–5.2) 4.7±0.3 (4.1–5.3) c 16.13 ± 3.86(13,8-26,7) 15.8 15.9±1.1 (13.5–17.3) 16.8±1.1 (14.6–18.6) 16.5±1.1 (15.3–19.6) c’ 2.29 ± 0.38(1,48-2,97) 2.7 2.6±0.2 (2.3–3.1) 2.7±0.2 (2.3–3.0) 2.6±0.2 (2.3–3.0) V (%) 72.84 ± 1.97(70-77) 71.9 72.8±1.1 (70.7–74.9) 73.2±0.8 (71.5–74.4) 70.6±1.2 (68.9–72.8) Stylet length 16.074 ± 0.74(15-17,4) 17.8 17.3±0.4 (16.7–18.1) 17.7±0.4 (17.0–18.5) 18.3±0.3 (17.7–19.2) Stylet shaft length 8.01 ± 0.54(7,1-9) 9.1 9.1±0.3 (8.3–9.7) 9.5±0.3 (8.6–10.0) 9.8±0.2 (9.5–10.5) Stylet knob width 3.88 ± 0.43(3,4-4,9) 4.1 4.1±0.2 (3.9–4.5) 4.3±0.3 (3.9–4.7) 4.2±0.2 (3.8–4.6) Stylet knob height 1.94 ± 0.26(1,4-2,4) 2.0 2.3±0.2 (2.0–2.5) 2.3±0.2 (2.0–2.7) 2.4±0.1 (2.1–2.6) DGO from stylet base 2.66 ± 0.49(1,8-3,3) 2.8 3.1±0.3 (2.5–3.7) 3.0±0.3 (2.6–3.4) 3.1±0.3 (2.6–3.6) Lip width 8.02 ± 0.43(7,2-8,8) 8.8 8.6±0.4 (8–9.2) 8.6±0.3 (8.0–9.1) 8.6±0.3 (7.8–9.1) Lip height 3.31 ± 0.32(2,9-4) 2.5 2.9±0.2 (2.5–3.2) 2.5±0.2 (2.2–3.0) 2.8±0.2 (2.5–3.4) Centre of metacorpus 48.96 ± 3.31(44-54) 56.3 60.3±3.5 (51.8–67) 56.3±2.6 (51.5–62.2) 60.1±2.0 (54.7–64.6) Cardia 71.91 ± 5.98(63-83,8) 83.7 92.6±5.1 (83.5–102.5) 89.9±3.5 (85.1–99.0) 92.8±3.7 (85.9–101.6) End of pharyngeal gland Lobe 106.48 ± 11.59(86-120) 118.8 129.9±5.9 (117.2–139.3) 139.8±9.4 (122.7–158.1) 137.3±7.5 (125.0–158.1) Secretory/excretory pore 65.33 ± 7.79(48-75) 80.2 83.5±4.2 (76.1–90.3) 93.8±5.6 (82.4–103.4) 92.7±4.6 (78.2–99.3) Pharyngeal overlap 38.08 ± 9.11(26,9-58) 35.0 37.8±4.4 (29.4–44.4) 50.4±8.8 (31.4–65.6) 44.4±7.2 (33.5–66.4) Median bulb length 10.49 ± 1.12(9-12) 13.9 13.7±0.8 (12.8–15.3) 13.5±0.6 (12.6–14.7) 14.7±0.7 (13.9–15.8) Median bulb width 8.17 ± 0.43(7,6-8,8) 10.2 10.4±0.9 (9.1–11.8) 10.8±0.4 (10–11.3) 11.1±0.4 (10.8–11.9) E.P. (%) 14,5± 2,4(10-18) 14.2 14.3±0.8 (12.9–15.6) 15.0±0.5 (14.0–16.1) 14.3±0.7 (12.4–15.5) Max. body diam. 21.20 ± 2.55(14,9-24,3) 24.4 23.7±2.1 (19.3–27.4) 21.1±1.3 (18.7–23.8) 24.2±1.7 (21.0–26.2) Vulval body diam. 17.68 ± 2.32(12,8-21,3) 20.3 22±1.4 (19.8–24.4) 20.5±1.3 (18.2–23.4) 23.0±1.6 (20.1–25.6) Anal body diam. 12.67 ± 1.70(10-15) 13.0 14.1±1.4 (12.2–16.2) 14.1±0.7 (13.0–15.3) 15.3±0.7 (13.7–16.1) Anterior genital tract length 169.11 ± 34.38(123-240) 176.1 177.1±38.1 (117.7–262.9) 145.3±20.4 (114.6– 165.2±27.2 (132.5–242.9) 204.0) Tail length 28.98 ± 5.37(18,8-36,3) 35.5 36.6±2.8 (31.5–41.6) 37.6±2.2 (33.2–41.9) 39.5±2.6 (34.6–43.7) No. of tail annuli 27.3 ± 2.94(23-32) 25 24.6±2.4 (22–28) 29±2 (25–33) 32±3 (25–36) Vulva to anus distance 94.18 ± 15.37(62-114) 124.8 120.9±10.8 (107.1–152.3) 130.3±11.8 (105.7– 152.1±12.4 (134.4–176.2) 151.3) Post-uterine sac 34.18 ± 15.36(25-45) 37.6 41.3±5.6 (35.5–53) 39.5±3.8 (32.5–49.0) 49.5±5.9 (41.8–60.6) Lateral field width 4.81 ± 0.99 (3-6) 6.1 6±0.6 (5.1–7.1) 6.0±0.6 (5.2–7.3) 6.8±1.0 (5.7–7.6) Phasimids from tail terminus 17.52 ± 2.14(13,2-20) 19.8 17.7±2.1 (15.2–20.5) 18.9±2.4 (16.2–23.8) 20.5±1.2 (18.9–23.9)

Pratylenchus zeae Graham, 1951 Morphological characterization Body slim, generally straight after glycerin fixation; with faint annuli. Lateral field with 4 incisures extending along tail beyond phasmids, occupies 19% of corresponding body diameter. Lip region not set off from body, bearing 3-4 annuli. Labial framework heavily sclerotized extend into body about one annule. Stylet with round, anteriorly flattened basal knobs. Dorsal pharyngeal gland opening at 2,73± 0.53 μm posterior to the stylet base. Secretory-excretory pore at 15,36% of the body length from the anterior end. Hemizonid about 2 body annuli long, before the excretory pore. Pharyngeal gland lobe ventrally overlapping intestine. spermatheca around, small, without sperms. Phasmids slightly posterior to middle of tail. Tail tapering, with 22-29 annuli, terminus variable, pointed, narrowly rounded to subacute.

Molecular characterization Five sequences of 446 bp length of the partial 18S rRNA products were obtained, based on DNA template of five single females recovered from five rice fields in Haiti. The Intraspecific variations of the 18S sequences obtained for this population of Pratylenchus zeae were 0,0-0,2% (0 - 8 bp). A Blastn search of the generated sequences revealed 98,72-99,19% similarity with the deposited sequences of Pratylenchus zeae into GeneBank (KY424132, KY424132). The inter-specific variation of the Partial 18S rRNA gene between the obtained sequences and the deposited sequences of P. zeae into GeneBank were 0,00 - 3% (0-13 bp). The Baysian inference phylogenetic tree (Fig. 7-A) based on the 18S rRNA gene; from a multiple alignment of 445 characters, placed the P. zeae from Haiti amongst the other sequences of P. zeae deposited into GeneBank in a maximally supported clade. From the collected population of Pratylenchus zeae of this study, four sequences of 390-bp were obtained, based on DNA template of four single females from 4 different fields. The Intraspecific variations of the COI sequences obtained for this population of P. zeae were 0,0 – 0,19% (1 bp). A Blastn search of the generated sequences revealed 99,18-100,0% similarity with the deposited sequences of P. zeae into GenBank (KP903440, KF765435). The inter-specific variation of the COI gene between the obtained sequences and the deposited sequences of P. zeae into GeneBank were 0,0 – 1% (0-4 bp). The Baysian inference phylogenetic tree (Fig. 7-B) based on COI gene; from a multiple alignment of 390 characters, placed the P. zeae from Haiti amongst the other sequences of P. zeae deposited into GeneBank in a maximally supported clade. Remarks A comparison with the species type and two other populations described by Wang et al. (2015) which are all specimens collected from China, revealed that the morphology and the morphometric data ( Table 5) of the populations recovered from maize in Haiti are congruent with these references. While the allometric parameters of the De Mann of the Haitian populations are well matched with the other populations: The specimens recovered have a smaller body size, more than 100 µm less compare to the average body length of the other populations described by Wang et al. (2015), including the holotype and the paratype. Consequently, the Haitian population have a smaller size for most of the features measured, perhaps the position of the vulva, the lip width and the position of the phasmid from the tail terminus presented very minor difference compared to the Chinese population. The molecular data from two different genes combined with the phylogenetic relationship analysis from the Baysian inference tree (Fig. 10) confirmed the Pratylenchus populations of Haiti presented in this study belong to Pratylenchus parazeae. The morphological and the morphometrical (Table 6) analysis of the population of Pratylenchus zeae recovered from rice in Haiti revealed full agreement with the morphometrics data from the original description of this specie (Graham, 1951). Although, the Haitian population have a smaller body size that lower the morphometrics value compare to other populations described from other regions in the world where the average body size exceed often 500 µm, 540 ± 5,4 µm (Roman &Hirschmann, 1969); 504 ± 37,9 µm (Tatiana and al, 2016). However, the position of the vulva and the stylet showed few variations compared with the populations presented in the table 6. The phylogenetic analysis confirmed the belongness of this Pratylenchus population from rice fields in Haiti to Pratylenchus zeae. The morphological and morphometrical comparison of the specimens of Pratylenchus zeae and Pratylenchus parazeae recovered from this presented study, underlined the difficulty to segregate morphologically the two species, they are morphometrically cross overlapping. The total body length, an important feature presented by Wang et al (2015) to differentiate the two species, is rejected with the

findings on the haitian populations of P. parazeae. The body length of the specimens measured compared to the paratype ((453.5 ± 53,08 (363-509) vs 587.5±37.8 (527.8–682.6)) are significantly low. The maximum body length of the Haitian population is lower the inferior limit given for the paratype and the two subsequent populations presented by Wang et al (2015). The body length of the Haitian population of P. parazeae and most of the subsequent features measured for morphometrical characterization fall in the morphometrical characterization of P. zeae. Chen and al(2009) in Taiwan have identified a Pratylenchus population as P. zeae (L= 520-660 μm) and deposited the sequence into genebank (J643590). However, the The Baysian inference tree presented by Wang et al (2015) for the ITS region, placed the P. zeae of Taiwan within a maximally supported monophyletic clade of P. parazeae. The molecular analyse confirmed that the population of Chen and al(2009) morphologically characterized as P. zeae are molecularly belong to P. parazeae. Based on the observation on the haitian population of Pratylenchus zeae and Pratylenchus parazeae , and the mis-characterization of Pratylenchus zeae by Chen and al(2009), we suggest the consideration of P. parazeae as a cryptic of P. zeae instead of a member of the species complex of P. zeae.

A: 18S

Fig. 8. Phylogenetic trees of Pratylenchus species based on 18S rDNA data (A), D2D3 LSU rDNA data (B) and ITS1-5.8- ITS2 rDNA data (C) obtained from Bayesian inference (BI) performed with MrBayes v3.1.2 using a GTR + I + G model. Branch support is indicated with PP: high (≥95% PP)/ medium (>95% to ≥ 50% PP)/ low (<50% PP).

B: 28S

C: COI

Table 6. Measurements of females of Pratylenchus zeae on rice in Haiti. All measurements are in µm (except for ratio) and in form: mean ± SD (range)

(Graham T. (Merny, (Roman &Hirschmann, (Tatiana and al, 2016) Present study W., 1951) 1970) 1969) Costa-Rica Crops Maize Rice Maize Rice n - 25 50 20 10 L (µm) 360-580 340-550 540 ± 5,4 (453-667) 504 ± 37,9 (434-556) 387 ±52,3(352-500) a 25-30 22-33 27.2 ± 0,35 (20.5-32.8) 25.8±1.8 (23.1–29.8) 17,2±7,02(17,9-20,9) b 5.4-8.0 3,3-4,9 6.5 ± 0,06 (5.5-7.9) - 5,513±1,09 (4,19-6,84) b' - - - 4.1±0.5 (3.2–4.7) 3,91±0,65 (3,06-4,79) c 17-21 13-18 15.2 ± 0,18 (13.0-17.7) 17.4±4.0 (12.5–23.9) 13,44±1,1 (12,2-14,91) c' - - - 2.4±0.4 (1.5–3.1) 2,64±0,35 (2,38-2,89) V% 68-76 69-74 70.9 ± 0,15 (69.0-75.0) 71.1±1.8 (67.3–74.4) 69,76±2,8 (67-75) Stylet (µm) 15-17 15-18 15.5 ± 0,06 (13.6-16.6) 15.0±0.6 (14.0–16.0) 15,1±0,46(14,6-17) DGO (µm) - - 2.4 ± 0,03 (1.8-3.0) - 2,73(1,6- 3,72) S-E pore - - 88.1 ± 0,9 (74.8-104.4) 81.4±6.7 (65.0–95.3) 59,48±7,23(48,4-68) Lip height - - 2.45 ± 0,02 (2.4-3.0) - 2,87±0,32(2,5-3,12) Lip width - - 7.81 ± 0,03 (7.2-8.4) - 8,34±0,42(7,9-8,64) Max. body - - 19,8 ± 0,33 (16-24) 33.2±3.0 (28.9–38.2) 18,86±3,2(13-23,9) diam. Tail length - - 35.6 ± 0,4 (24-40) 27.3±5.8 (21.0–33.0) 28,9±3,24(25-34,5) Phasmids from - - 19,16 ±0,25 (16-22) - 13±0,56(12,6-13,4) tail terminus

Table 7. Measurements of juveniles of Meloidogyne graminicola on rice in Haiti and other populations. All measurements are in µm (except for the ratio) and in form: mean ± SD (range)

Character Present study (Golden and Birchfield, (Saha et al, 2017) (SALALIA et al., 2017) 1965) n 17 - - 174 L 440 ±51,36 (330-504) 441(415-484) 468.4 (395.0-575.6) 483 (408,8 – 608) a 29,06 ±6,68 (20,18-42) 24.8(22.3-27.3) 31.6 (22.5-41.5) 33,42 (21,8 – 44,5) b 5,97 ±0,29(5,6-6,3) 3.2(2.9-4.0) 2.8 (2.2-4.2) 5,85 (4,4 – 8,6) b’ 3,76 ±0,78(2,9-2,4) - - 3,18 (2,0 – 4,7) c 6,51 ±1,31(5,5-7,4) 6.2(5.5-6.7) 6.9 (4.9-8.7) 6,4 (5,4 – 8,3) c’ 5,8 ±0,48(4,1-6,2) - - 7,2 (5,0 – 9,0) DGO 3,2 ±0,56(2,4-4,7) 2.8-3.4 3.7 ± 0.6 (2.6-4.5) 3,32 (2,0 – 4,5) Stylet length 12 ±1,21(10,3-13,98) 11-12 10.7 (9.2-13.4) 11 (10 – 12,5) Pharynx length 96,2 ±24,6(70-130) - - 158,8 (100 – 249) Cardia 77,6±12,6 (58-90) - - 83,76 (64-121) Excretory pore 65,9 ±8,2(57,3-76,4) - - 80,3 (65 – 105) Max. body diam. 15,9±.2,6(11,3-18,9) - - 14,4 (12 – 17) Diam. at anus 8,7±2,3(5,8-11) - - 10,5 (9 – 13) Tail length 43,4±12,7(31,8-64) 67-76 69.2 (50.0-82.2) 76 (55 – 97) Hyalin 23,9±2,1(19,5-26) 14-21 22.0 (15.6-27.4) 22,2 (13 – 35)

Fig. 9. Pratylenchus parazeae. LM of female. A,C,D,E,F: tail region; B: Anterior region; G: Vulva region; H: Entire female (Calibration: A - F= 100 µm; G= 40 µm; E= 10 µm).

Fig.10. Pratylenchus zeae. LM of female. A, E,F,G, H,I: tail terminus; B: Anterior region; C: Entire female; D: Vulva region (Calibration: A,B, D, E,F,G, H,C= 10 µm).

Meloidogyne graminicola Golden and Birchfield, 1965 Morphological characterization of the second stage juvenile Body vermiform, tapering at both extremities but much more so posteriorly. Head not offset, with weak cephalic framework, bearing 3 annuli. Body cuticular annulation fine but willingly visible. Lateral fields with 4 incisures, outer incisures finely crenated, occupying 1/4 of body width near middle. Stylet slender, knobs sloping rounded. Orifice of dorsal oesophageal gland 3,2 ±0,56 μm from the stylet knobs. Excretory pore at 15% of the body length from the anterior end. Metacarpus well develop, oval to spherical. Tail filiform with hyaline portion. Tail terminus rounded, slightly clavate. Molecular characterization Phylogenetic analysis has been done on seven partial 18S rRNA sequences (725 bp), obtained from DNA template of seven juveniles from four rice fields in Haiti. The obtained sequences were similar 98,5 to 99,55% to the deposited sequence (MG273438, MK301102, MK301101) of M. graminicola into Genebank. The intra-specific variations between the obtained sequences and the inter-specific variations between the generated sequences and the existing sequences of M. graminicola were respectively 0,0-0,7 % (0- 5bp) and 0,0-5% (0-36 bp). The Baysian inference tree placed the 18S sequences of the specimens from Haiti in a maximally supported clade together with sequences of M. graminicola, M. oryzae, M krali and M. naasi. Two sequences of 830 bp were obtained for the D2-D3 of the 28S rRNA gene. The generated sequences were similar 98,2 to 99,20% to the deposited sequences of M. graminicola into Genebank (KY660545, KF751067, MH359152). The intra specific variations between the obtained sequences and the inter- specific variations between the generated sequences and the existing sequences of M. graminicola were respectively 2 % (17 bp) and 0,0-3% (0-24 bp). The Baysian inference tree placed the 28S sequences of the specimens from Haiti together with subsequent sequences of M. graminicola and a sequence of M. salasi (KY962665) in a Maximally supported clade. Remarks Morphologically and morphometrically ( Table 7), our population of Meloidogyne graminicola aligned with the description of the type specimens ( Golden and Birchfield, 1965). The body size of the Haitian population have a smaller tendency compared to the findings of Saha et al (2017) and Salalia et al (2017). The D2-D3 of the 28S rRNA gene is unresolved for species delimitation between M. graminicola and M. salasi. Therefore, the two species formed a maximally supported monophyletic clade in a Baysian inference tree. However, M. graminicola and M. salasi are well delimited based on the ITS gene with more 2% dissimilarity (KY962656). The 18S gene is unresolved for species delimitation between M. graminicola, M. krali, M. naasi and M. oryzeae.

A: 28S

B: 18S

Fig.11. Phylogenetic trees of Meloidogyne species based D2D3 28S rDNA data (A) and 18S rDNA data (B) obtained from Bayesian inference (BI) performed with MrBayes v3.1.2 using a GTR + I + G model. Branch support is indicated with PP: high (≥95% PP)/ medium (>95% to ≥ 50% PP)/ low (<50% PP).

Fig.12. Meloidogyne graminicola. LM of juveniles and males. A: tail region of juvenile; B: Lateral lines of on juvenile body; C: Anterior region juvenile; D: Entire juvenile; E: Anterior region male; F: Lateral view tail region male; G: Lateral lines male; Ventral view tail region male (Calibration: A= 20 µm; B, C, E,F,G= 100 µm; D= 10 µm).

Helicotylenchus eritrynae Zimmermann, 1904 Morphological characterization of Helicotylenchus eritrynae Female body C-shaped to spiral. Lip region anteriorly rounded, not offset, bearing 5-6 annuli. Stylet robust, with shaft portion 56,4% of the stylet length. Stylet knobs; anterior faces flattened, indented or flattened and inclined backwards. Orifice of dorsal oesophageal gland 6,47±3,88 μm from the stylet knobs. Median bulb oval to round, at 10% of the body length from the anterior end. Pharynx with a ventral overlap gland. Excretory pore. at 15,4% of total body length. Hemizonid two to three annuli long, located one to two to annuli anterior to excretory pore. Two branches of the reproductive system both functional, anterior branch. Spermatheca set off, filled with sperm. Lateral field 6,29±0,61 µm wide; crenate and incompletely areolated anterior and posterior to vulva. Tail with seven to ten ventral annuli; tail more curved dorsally with a rounded or pointed annulated ventral projection, mucro seen. Molecular characterization of Helicotylenchus eritrynae Partial 18S rRNA Three 633-bp of the partial SSU products were sequenced, based on DNA template of four single females from four rice fields. Intraspecific variations of theses sequences recovered from this population of Helicotylenchus erytrinae were 0.0–1.2 % (0–9 bp). Based on nucleotides differences, Helicotylenchus erytrinae is closely related to H. crenacauda (16 bp), H. indicus (17 bp), H. dihystera (19 bp), H. pseudorobustus (22 bp). The sequence of Helicotylenchus erytrinae formed a maximally supported (100%) monophyletic clade in the Baysian inference tree. This study provided the first sequences of Helicotylenchus eritrynae for the partial SSU 18S rRNA. D2-D3 of the 28S rRNA Two 600 bp of the 28S rRNA SSU products were sequenced, based on DNA template of two single females from two rice fields. The Intraspecific variations of the 28S sequences obtained for this population of Helicotylenchus erytrinae were 0,0-0.5% (0 - 3 bp). Based on nucleotides differences, Helicotylenchus erytrinae is closely related to H. paraplatyurus (25 bp), Pseudorobustus (28 bp), H. broadbalkiensis (27bp). The Baysian inference phylogenetic tree (Fig. 17) based on the 28S rRNA gene; from a multiple alignment of 600 total characters, showed Helicotylenchus erytrinae formed a maximally supported monophyletic clade. This study provided the first sequences of Helicotylenchus eritrynae for the D2-D3 28S rRNA gene. Remarks The morphological and the morphometrics data (Table 8) of this Helicotylenchus population agree with the original description (Zimmermann, 1904) and with morphometrical characteristics of H. erythrinae from other places in the world (Marais, 2001). Therefore, we confirmed, this population of Helicotylenchus belong to H. erythrinae. As the first sequences are provided by this study, no molecular comparisons are possible at this time.

Table 8. Measurements of females of Helicotylenchus erithrynae on rice in Haiti and other populations. All measurements are in µm (except for ratio) and in form: mean ± SD (range)

Present study Topotype Topotype French Guiana Guadeloupe female female n 10 4 20 38 18 L 636,6±38,8(566-682) 0,48 -0,61 520-710 649 ±8904 (463-797) 649±6404 (547-760) a 25,26±1,96(22,09-29,28) 23-26 27-33 25.6± 2.7 (19.5-29.5) 26.4 ±2.7 (22.3-30.8) b 7,72±0,62(6,86-8,65) 5-5,9 5,3-6,7 5.3 ± 0.5 (404-5.8) 5.3 ±004 (4.8-604) b’ 5,43±0,28 (5,0-5,77) 3,8-4,7 4,0-5,5 4.6± 004 (3.8-5.0) 4.8 ±004 (3.8-504) c 45,13±6,92(31,6-53,9) 27-34 27-38 33.4 ± 4.9 (24-46.8) 29.2 ±2.8 (23.3-33.5) c’ 1,06±0,17(0,9-1,151) 1,0-1,6 1,1-1,7 1.3 ±0.2 (1.0-1.8) 1.5±0.2 (1.3-1.9) V (%) 60,20±1,32(57,42-62,03) 60,0-65,0 59-65 62 ±1.6 (59-65) 63 ±1.8 (60-66) Stylet length 22,5±0,66(21-23,6) 23,0-26,0 23-28 24.1:t 1.6 (21-28) 25 ± 1.2 (23-28) Stylet shaft length 12,7±0,70(11,56-14) - - - - Stylet knob width 3,94±0,36(3,42-4,6) - - - - Stylet knob height 1,99±0,22(1,7-2,3) - - - - DGO from stylet base 6,47±3,88(3-11,95) - - 17 ±4.2 (8-23 11 ±1.1 (9-12) Lip width 6,96±0,35(6,7-7,8) - - - - Lip height 3,44±0,2(3,04—3,8) - - - - m 45,44±2,51(39,13-48,16) 48-50 46-50 47±1.8 (44-53) 50±1.5 (47-53) O 28,54±16,79(13-53) 33-44 25,39 68±15.3 (36-90 43 ±404 (33-51) Centre of metacorpus 63,59±1,33(61-66) - - - - Cardia 82,7±5,14(73-90) - - - - End of pharyngeal gland Lobe 117,09±3,4(110-120) - - - - Secretory/excretory pore 98±3,31(94-103,32) 73 ±4.0 (63-84 73 ±6.2 (55-82) Pharyngeal overlap 38,43±4,86(35-44) - - - - Median bulb length 11,12±0,57(10,13-12) - - - - Median bulb width 8,68±0,92(7-10) - - - - S-E P 98±3,31(94-103) - - - - Max. body diam. 25,29±1,84(21-27,82) - - - - Vulval body diam. 23,65±1,16(22,4-25,62) - - - - Anal body diam. 13,6±0,66(12,3-14,53) - - - - Anterior genital tract length 195,5±4,94(192-199) - - - - Tail length 14,43±2,56(11,16-20) - - - - No. of tail annuli 7,6±1,17(7-10) - - - - Post-uterine sac 136,66±35,83(111-162) - - - - Lateral field width 6,29±0,61(5-7) - - - - Phasmids from tail terminus 18,81±2,85(13-24) - - - -

Fig.13. Phylogenetic trees of Helicotylenchus species based on D2-D3 of the 28S rRNA data obtained from Bayesian inference (BI) performed with MrBayes v3.1.2 using a GTR + I + G model. Branch support is indicated with PP: high (≥95% PP)/ medium (>95% to ≥ 50% PP)/ low (<50% PP).

Fig.14. Helicotylenchus erithrynae. LM of females. A: Entire female; B: Anterior region; C: Cephalic region; D: Vulva region; E: Lateral lines; F - I: Tail terminus (scale bars? A= 20 µm; B, C, D, E, F, G, H, I= 100 µm).

Helicotylenchus retusus Siddiqi & Brown, 1964

Morphological characterization Female body generally C-shape to rarely spiral. Lip region anteriorly flattened not set off, with no annuli. Stylet knobs anterior faces indented. Orifice of dorsal oesophageal gland 11,09±2,36 µm from the stylet knobs. Median bulb oval to spherical. Pharyngeal gland ventrally overlapped the intestine. Excretory pore at 14% of the body length from the anterior end. Hemizonid two to three annuli long, located one to two annuli anterior to excretory pore. Annuli at midbody width of 1,1±0,01 µm. Vulva post median from the anterior end. Two branches of reproductive system, both functional. Spermatheca off-set, thick-walled and empty. Lateral field 4,58±0,78 µm wide. Phasmids located ten to fourteen annuli anterior to anus. Rectum does not overlap anus. Tail ventrally curved, with rounded end, with nine to fifteen ventral annuli. Male Not found. Molecular characterization of Helicotylenchus retusus Partial 18S rRNA Sequences for the partial SSU 18S gene have been obtained from DNA template of single females recovered from rice fields in Haiti. Intraspecific variations of theses sequences recovered from this population of Helicotylenchus retusus were 0.3–0.8 % (2–5 bp). Based on nucleotides differences, Helicotylenchus retusus is closely related to H. erythrinae (64 bp), H. indicus (76 bp), H. crenacauda (87 bp). The sequence of Helicotylenchus retusus formed a well support monophyletic group in the Baysian inference tree. This study provided the first sequences of Helicotylenchus retusus for the partial SSU 18S rRNA gene. D2-D3 of the 28S rRNA Two 633 bp of the 28S rRNA SSU products were sequenced, based on DNA template of two single females from two rice fields. Intraspecific variations of theses sequences recovered from this population of Helicotylenchus retusus were 0.6 % (4 bp). Based on nucleotides differences, Helicotylenchus retusus is closely related to H. paraplatyurus (20 bp), H. microlobus (21 bp), H. Pseudorobustus (26 bp), H. digonicus (24 bp). The Baysian inference phylogenetic tree based on the 28S rRNA, from a multiple alignment of 620 total characters, placed Helicotylenchus retusus in a maximally supported (100%) monophyletic clade. Remarks on Helicotylenchus retusus The morphological and the morphometrical data (Table 9) recovered from this population of Helicotylenchus matched very well with the description of the type population by Siddiqi & Brown (1964) and the re-description of Sher (1966) of Helicotylenchus retusus. Therefore, we affirmed the belonging of these specimens to Helicotylenchus retusus. Although, the Haitian population have a smaller body size (xx vs xx) compared to the type description and the re-description. The vulva of the specimen recovered is also more posterior 64% vs 62% compare to the paratype of Sher (1966). Considering the description and the redescription of Helicotylenchus retusus, there is a discrepancy between the shape of the tail illustrated by siddiqi & Brown (1964) and the one illustrated by Sher (1966): respectively tail terminus obtusely rounded, sub-conoid versus symmetrical hemispherical. According to Marais (2001), Sher (1966) used the type material deposited in the nematode collection of Rothamsted and the collections of Brown & Siddiqi for the re-description of this species. Therefore, the tail shape described by Sher (1966) appeared

to be the correct one. The specimens recovered from the rice fields in Haiti have all a hemispherical tail terminus and aligned 100% with the re-description of Sher (1966).

Table 9. Measurements of females of Helicotylenchus retusus on rice in Haiti. All measurements are in µm (except for ratio) and in form: mean ± SD (range)

(Siddiqi & (Siddiqi & Brown, 1964) (Sher ,1966) Martinique Present study Brown, 1964) Paratype Paratype Holotype n 1 5 1 12 10 L 750 730-770 750 788±94.2 (702-934) 725±61,5(663-880) a 33 33-36 34 27.7±2.0 (2.3-30.7) 31,69±2,0(29,33-35,79) b 6 5,7-6 5,8 5.9 7,61±0,86(6,57-8,82) b’ 4,7 6.1 ±0.3 (5.7-6.5) 6,66±0,65(5,8-7,61) c 53 48-53 52 50.4±5.0 (43.1-58.7) 57,48±5,81(49,31-67,5) c’ - - 1,0 0.9±0.1 (0.8-1.1) 1,15±0,12(1,0-1,33) V (%) 64 61-64 62 62 ±0.6 (61-63) 64,45±1,95(61,5-67,90) Stylet length 26-27 27 24 ±1.3 (22-26) 28,46±0,71(27-29,41) Stylet shaft length - - - 15,69±064(14,5-16,57) Stylet knob width - - - 4,2±0,32(3,7-4,66) Stylet knob height - - - 2,23±0,29(2-2,8) DGO from stylet base - - - 12±1.0 (10-13) 11,09±2,36(6-13,5) Lip width - - - 6,62±0,58(5,3-7,35) Lip height - - - 3,82±0,36(3,21-4,2) m - - 46 47±1.9 (42-49) 44,87±1,95(41,6-47,39) O 44-48 42 50 ±:3.8 (41-55) 38,98±8,37(21,42-50) Centre of metacorpus - - - - 63,2±7,98(53-75) Cardia - - - - 95,64±6,85(84-103) End of pharyngeal gland - - - - 109,14±7,75(96-122) Secretory/excretory pore - - - - 101,9±8,12(89-114) Pharyngeal overlap - - - - 26,5±3,70(18-30) Median bulb length - - - - 10,17±0,77(8,5-11) Median bulb width - - - - 8,02±1,03(6,5-10,21) Max. body diam. - - - - 22,98±2,64(18,8-28,78) Vulval body diam. - - - - 20,75±2,69(17-26) Anal body diam. - - - - 17,12±1,54(15,5-20) Anterior genital tract length - - - - 259,33±59,23(156-330) Tail length - - - - 12,74±1,81(10-15,04) No. of tail annuli - - - - 11,22±2,63(9-15) Post-uterine sac - - - - 158,35±34,55(100-210) Lateral field width - - - - 4,58±0,78(4-5,87) Phasimids from tail - - - - 27,75±2,63(25-31,87) terminus

Fig.15. Helicotylenchus retusus. LM of females. A: Anterior region; B: Phasmid; C, E:Tail region; D: Entire female body; F: Lateral lines on mid-body; G: Vulva region (Calibration: D = 20 µm; B, C, E, F, G= 100 µm).

Quinisulcius acutus Allen, 1955

Morphological characterization Female body C-shape to spiral, tapering at both ends. Labial framework slightly offset, moderately sclerotized, broadly rounded; bearing six annuli. stylet with prominent stylet knobs, sloping posteriorly. Median bulb oval spherical at 10% of the total body length from the anterior end. Excretory pore at 18% from the anterior end. Pharyngo-intestinal valve inconspicuous, cardia distinct. Lateral field more than ¼ of the body diameter wide and hearing five incisures; the outer most incisures crenate, partly non areolated at the tail. Hemizonid 2 annules anterior to pore. Deirids about opposite pore. Vulva post median, from the anterior end. Female reproductive system double opposed; spermatheca small, empty. vagina width less than one-half of body width. Phasmid located at 31% of the tail length from the anus. Tail ventrally curved, conoid, with about 17-20 annules; terminus bluntly, pointed, without annuli or striations.

Molecular characterization of Quinisulcius acutus Partial 18S rRNA Five sequences (906 bp) have been obtained for the partial SSU 18S rRNA, based on DNA template of five single females from two different populations in rice fields in Haiti. Intraspecific variations of theses sequences recovered from these populations of Quinisulcius acutus were 0.0–3.1 % (0–28 bp). Based on nucleotides differences, Quinisulcius acutus is closely related to Tylenchorhynchus clarus (42 bp), Tylenchorhynchus microconus (40 bp), Tylenchorhynchus claytoni (42 bp), Bitylenchus mediterraneus (45 bp). The Baysian inference phylogenetic tree (Fig. 20: A) based on the partial SSU 18S rRNA from a multiple alignment of 861 bp, placed the sequences of Quinisulcius acutus in a maximally supported clade and was sister clade and was sister with a fully supported Tylenchorhynchus clade containing (T. claytoni, T. microconus, T. clarus, T. aduncus).

D2-D3 of the 28S rRNA Three sequences (804 bp) of the D2-D3 28S rRNA region were obtained from the populations of Quinisulcius acutus of Haiti. The inter-specific variation between the obtained sequences varied from 0.0- 0,8 % (6 bp). Based on nucleotides differences, Quinisulcius acutus is closely related to Neodolichorhynchus estherae (93 bp), Neodolichorhynchus phaseoli (100 bp), Bitylenchus maximus (101 bp), Bitylenchus mediterraneus (107 bp). The Baysian inference phylogenetic tree (Fig. 20: B) on the 28S rRNA, from a multiple alignment of 789 characters, placed Quinisulcius acutus in a maximally supported monophyletic clade and was sister taxon with a fully supported clade of Neodolichorhynchus containing (N. estherae and N. phaseoli).

Table 10. Measurements of females of Quinisulcius acutus on rice in Haiti. All measurements are in µm (except for ratio) and in form: mean ± SD (range)

(Allen, 1955) (Siddiqi, 1971) Present study type females n 1 - 5 L 600-750 650-700 508±19,79(494-522) a 31-38 27,9-35 24,77±0,1(24,7-24,85) b - 4,8-5,3 4,46±0,07(4,41-4,51) b’ - - 4,25±0,03(4,22-4,25) c 17-23 15,8-19,3 15,48±1,39(14,5-16,46) c’ 2-2,2 2,2-2,8 2,34± 0,35(2,08-2,59) V (%) 56-58 55,8-58 56±0,09(55,93-56,07) Stylet length 15-17 16-17 14,15±0,21(14-14,3) Lip width - - 6,66±0,2(6,52-6,8) Lip height - - 3,15±0,21(3-3,3) Cardia - - 113,83±2,59(112-115,67) End of pharyngeal gland Lobe 119 - 119,5±3,53(117-122) Secretory/excretory pore - - 91,7±7,7(86-97) Centre of metacorpus - - 50,05±0,07(50-50,11) Median bulb length - - 10,61±2,28(9-12,23) Median bulb width - - 8,30±1,56(7,2-9,41) Max. body diam. 20 - 20,5±0,70(20-21) Vulval body diam. - - 18,25±2,47(16,5-20) Anal body diam. - - 14,12±0,35(13,87-14,37) Anterior genital tract length - - 159 Tail length 30 - 33±4,24(30-36) No. of tail annuli 15-20 15-20 18,5±2,12(17-20) Post-uterine sac - - 112 Lateral field width - - 5,8 Phasimids from tail terminus - - 22±1,41(21-23)

ITS rRNA Two sequences (831-bp) of the ITS rRNA gene have been obtained from DNA template of two single females of Quinisulcius acutus from rice fields in Haiti. No Intraspecific variations has been observed between theses sequences. Based on nucleotides differences, Quinisulcius acutus is closely related to Paratrophorus bursifer (336 bp), Bitylenchus maximus (355 bp), Bitylenchus iphilus (357 bp). The two ITS rRNA sequences of Quinisulcius acutus formed a maximally supported (100%) monophyletic clade in the Baysian inference tree.

A= 18S

B= 28S

C= ITS

Fig.16. Phylogenetic trees of Dolichodorids species based on 18S rDNA data (A), D2D3 LSU rDNA data

(B) and ITS1-5.8-ITS2 rDNA data (C) obtained from Bayesian inference (BI) performed with MrBayes v3.1.2 using a GTR + I + G model. Branch support is indicated with PP: high (≥95% PP)/ medium (>95% to ≥ 50% PP)/ low (<50% PP).

Fig. 17. Quinisulcius acutus. LM of females. A: entire mature female; B: Anterior region mature female; C: Terminal bulb; D: Entire mature female; E: Cephalic annuli; F: Head region; G: Lateral lines at mid-body; H: Tail terminus; I: Vulva region. (Calibration: A, D = 40 µm; B, C, E, F, G, I= 100 µm).

Remarks on Quinisulcius acutus Morphological and morphometrical data of the specimens of Quinisulcius recovered from rice fields in Haiti were coincident with the original description (Allen, 1955) and the re-description of Siddiqi (1971). However, the population of Quinisulcius acutus of Haiti have some slight difference with the type specimens and the populations described by Siddiqi (1971) for the range of body length respectively ( 494- 522 vs 600-750) and (494-522 vs 650-700). Quinisulcius acutus have been reported in many places in the world (Ilker and al, 1998; Walters. and al, 1998) but no molecular characterization has been done for this species so far. The molecular analysis and the Baysian inference tree confirmed the fact that none sequences of the Quinisulcius genus have been yet submitted to Genebank. This study provided the first sequences (partial 18 SSU rRNA, D2-D3 of the 28S rRNA and ITS rRNA) for Quinisulcius acutus, the first also for this genus.

Mesocriconema sphaerocephalum Taylor,1936 Morphological characterization Female body ventrally arcuate. Cuticular retrorse, annuli with irregular margins. Body characterized by the presence of a high number of anastomoses throughout the body, forming a zig-zag pattern. The anterior body end appears truncated, labial disc low, submedian lobes and labial plates are small and not protruding. Stylet long and robust, stylet knobs anchor shaped. Typical criconematid pharynx. Secretory- excretory pores at 21 annuli from the anterior end. Vulva in the last quarter of the bbody from the anterior, open and simple with rounded lips. Gonad recurved. Vagina straight. Postvulval body part short with rounded tail. Males not found.

Molecular characterization Four sequences (794 bp) of the D2-D3 of the 28S region rRNA were obtained, based on DNA template of four single females recovered from cereal fields in Haiti. The 28S sequences obtained were similar 98,92- 99,02% to the deposited sequences of M. sphaerocephalum into Genebank. The intra-specific variation between the obtained sequences and the inter-specific variation between the obtained sequences and the deposited sequences of Mesocriconema sphaerocephalum into Genebank were respectively 1,5 -3% (12-25 bp) and 1,13-3,5 (9-28 bp). The single sequence (714 bp) obtained for the ITS rRNA gene was similar 90 % with a deposited sequences Mesocriconema sphaerocephalum into Genebank. The Baysian inference phylogenetic tree presented the 28S rRNA sequences of M. sphaerocephalum from this study together with the deposited sequences of M. sphaerocephalum into a maximally supported monophyletic clade ( ). The single ITS sequence obtained formed a maximally supported monophyletic clade with the other sequence of M. sphaerocephalum available into GeneBank.

Remarks Morphologically and morphometrically this population of Mesocriconema recovered from cereal fields in Haiti are congruent with the original description of Mesocriconema sphaerocephalum, Morphometrically, the Haitian population of Mesocriconema sphaerocephalum showed huge similarity to the population of Cost-Rica recovered from Mulberry trees (Peraza-Padilla, 2014), while it had a larger body size (306,25±1,76 vs 270±10 ) compared to the population of Venezuela recovered from sorghum fields (Renato, 2001).

Table 11. Measurements of females of Mesocriconema sphaerocephalum on sorghum in Haiti. All measurements are in µm (except for the ratio) and in form: mean ± SD (range)

Present study Taylor 1936, (Peraza-Padilla, 2014) (Renato, 2001) (Sorghum, Haiti) Loof and De (Mulberry trees, Costa Rica) (Sorghum, Venezuela) Grisse 1989 n 3 15 20 L 306,25±1,76 (305 – 307,5) 240-460 342.5 ± 33.7 (288.0-415.0) 270±10(259-282) a 11,35±0,66(10,89-11,82) - 9.3 ± 1.6 (6.4-12.6 7,3±0,6(7,8-8,9) b 3,10(3,10-3,11) - 3.6 ± 0.3 (3.1-4.1) 3,1±0,1(3,0-3,2) c 37,24±3,65(34,65-39,83) - - 47±1,9(49-50) c’ 0,34±0,01 (0,33-0,35) - - 0,3 VL/VB 0,79±0,17(0,67-0,92) 0,5-1 0.9 ± 0.2 (0.7-1.2) - V (%) 93,43±1,11(92,68-94,28) 90-97 92.0 ± 2.4 (84.5-94.2 95±1(93-96) Stylet length 49,5±0,70(49-50) 43-77 51.2 ± 10.1 (31.0-70.0) 49±2,6(44-50) Anterior end to end of 98,5±0,70(98-99) 96.6 ± 10.9 (78.0-110.0 88±1,4(86-90) pharynx s-excretory pore 100±1,41(99-101) - - 100±1(99-102) Max. body diam. 31 - 37.4 ± 4.7 (32.0-45.0) 32±1,5(31-35) Vulval body diam. 25,2±1,13(24,4-26) - 27.6 ± 2.8 (22.0-32.0 Anal body diam. 24±1,41(23-25) - - 16,8±0,3(16-17) Roes 20,5±0,7(20-21) - - 20-21 Rex 21 17-28 22.8 ± 2.2 (20.0-26.0 - Rv 4,5±0,7(4-5) 3-7 4.9 ± 0.8 (4.0-7.0 3-4 Ran 2 1-5 - 2 Rvan 2,5±0,7(2-3) 0-3 - 0-1 R 65±1,41(64-66) 55-82 66.1 ± 2.9 (61.0-71.0) 63-68

A: 18S

B: 28S

Fig. 18. Phylogenetic trees of Criconematids species based on D2D3 28S rDNA data (A) and ITS1-5.8- ITS2 rDNA data (B) obtained from Bayesian inference (BI) performed with MrBayes v3.1.2 using a GTR + I + G model. Branch support is indicated with PP: high (≥95% PP)/ medium (>95% to ≥ 50% PP)/ low (<50% PP).

Fig. 19. Mesocriconema sphaerocephalum. LM of females. A: entire mature female; B: Anterior region mature female; C: Anastomoses mid-body; D: Cephalic region; E: Tail region; F: Vulva region (Calibrationscale bars?: A = 40 µm; B, C, E, F= 100 µm).

Discussion Diversity and prevalence of PPN associated with the cereals in Haiti The prevalence and the density of plant-parasitic nematodes within the investigate cereal fields presented important variation according to the nematode species, crops and the ecosystems. Over the investigated fields, rice presented the highest nematode diversity compared to maize and sorghum. More than 90% of the PPN associated with the two other cereals are found to be associated with rice. This observation aligned with the publication of Sikora and al (2018) reporting that more than 200 nematodes species paratized on rice, around 100 species on maize and sorghum. In Haiti, rice is cultivated intensively while maize and sorghum are mainly cultivated extensively; associated with other crops in the fields. The high prevalence and density of plant parasitic nematodes on rice could be link to the production sytem, as Coyne and al (1999) and Gnamkoulamba and al. (2018) have demonstrated the system of production of rice (intensive system versus semi-intensive) influenced the prevalence and the density of PPN. The conventional agriculture (intensive system) have been demonstrated to increase the abundance of plant parasitic nematodes by accelerating their multiplication rate ( Landi et al, 2018). The high prevalence and the abundance of the Meloidogyne particularly M. graminicola, Pratylenchus zeae and the Helicotylenchus within the investigated rice fields confirmed the findings of many researchers assessing the diversity of plant nematodes associated with rice: Vietnam (Khuong, 1983), India (Jain et al., 2012; Sitaramiah, 1984), Nigeria (Babatola, 1984), United States (Smiley et al., 2004) , Kenya (Pili et al., 2016), Ghana (Coyne et al., 1999). The density of Meloidogyne graminicola and Pratylenchus zeae in the investigated fields have a lower tendency compared to the findings of the pre-cited authors. As example, according to Gilces et al. (2016), the density of Meloidogyne graminicola goes above 180 000 juveniles per 100 ml in some rice ecosystems in Ecuator, while the maximum density found in Haiti was 280 juveniles per 100 ml of soil. Fortuner et al. ( 1981) and Merny (1970) have noted that Prevalence and density of plant parasitic nematodes are driven by many biotic and abiotic factors, including the host status, nematode feeding specialisation, temperature and hydrology. In addition, the data will be influenced by a number of practical aspects such as sampling time and the relative extraction efficiencies for different genera (Coyne et al., 1999). Taken into account such conderations, the comparison of prevalence and density of nematodes from different agrosytems or even countries should be done cautiously and only the damage treshold of a particular specie in its ecosystem are reliable. The genus Helicotylenchus occured in all rice fields investigated. Over the nematodes recovered from the rice fields, five species of Helicotylenchus were identified including H. retusus the most abundant one. The high abundance and prevalence of the Helicotylenchus have been noticed also by Fortuner et al. ( 1981), Merny (1970) and Coyne et al. (1999). The genera Mesocriconema, Criconema, Quinisulcius, Rotylenchulus, Xiphinema and Tylenchorhynchus were identified in the investigated fields at low prevalence and relatively low density. These genera have been reported to have a low prevalence and low density in many rice ecosystems (Bridge et al., 1990; Babatola, 1984). Although, Bridge and al. (1990) suggests that the prevalence and the densities of these genera are not really link to the presence of rice in the field. The absence of Hirschmanniella sp. within the investigated rice fields differs this study from most of the studies conducted on rice’s nematodes ( Pascual et al., 2014; Khuong, 1983; Coyne et al., 1999). Maybe, the adoption of the improved system of production of rice in the late decennia, promoting intermittent irrigation instead of flooding could affect the prevalence and the density of the rice parasites of this genus. The Pratylenchus zeae group (P. zeae and P. parazeae) was the most prevalent and the most abundant nematodes species associated with maize in the investigated fields. Such results aligned with the affirmation of Sikora (2018) highlighting the Pratylenchus as the most prevalent and the most damageable nematodes of maize. The genus Helicotylenchus particularly H. erythrinae and Rotylenchulus reniformis have the maximum prevalence in the investigated maize fields, with important variation on the overall

45 density between the fields. This suggests that maize in Haiti is a very good host for the Rotylenchulus. The link of the Helicotylenchus species to a specific crop as parasite should be done with facts, considering their non-specifity in the feeding habit, their broad host range and their high tolerance to the abiotic soil factors (Coyne et al., 1999). The high prevalence of the genera Rotylenchulus and Helicotylenchus observed in this study agree with the findings of Jordaan et al. (1989) and Kagoda et al. (2010) . The criconematids (Criconema sp, Mesocriconema) and the dolichodorids (Quinisulcius acutus and Tylenchorhynchus agri) were more prevalent in the maize fields but with a lower density compared the rice fields. As Bridge and al. (1990) highlighted it above fore rice, the links between the population dynamic of these genera and the maize crops are not really clear also. The nematodes species recovered on maize in this study are congruent with the findings of many other studies about plant parasitic nematodes associated with maize in the world (Ikpeze, 2014; Mielie - Inligtingsgids, 2013; Sharma et al., 1993; Subedi, 2016).However, the absence of viable juveniles within the cyst of Heterodera consist a divergent point with some studies where the cysts nematodes were highlighted as major pest for maize (Aboul-Eid and al, 1981; Ringer and al 1987; Walter, 2016) .

Among the sorghum fields investigated, the Pratylenchus zeae group (P. zeae and P. parazeae) was the most prevalent nematode species. However, the density (per 100ml of soil) of the Pratylenchus in sorghum (70±36) is relatively lower compared to rice (99 ± 90) and maize (87±42). Pratylenchus zeae has been reported to be responsible of reduction of nutrients uptaked and root growth in sorghum (Koenning et al., 1999; Sikora and al (2018). However, taking account the low density observed within the investigated fields, phytopathologic test are required to determine the possibilities of harmfulness of P. zeae and P. parazeae on sorghum in Haiti. Other nematodes species like: Meloidogyne graminicola, Rotylenchulus reniformis, Mesocriconema sphaerocephalum, Criconema sp, Helicotylenchus erytrinae, Helicotylenchus sp. have been recorded with very low density in the investigated sorghum fields. The diversity of nematodes recovered from the sorghum fields in Haiti confirmed the findings of other researchers on sorghum nematodes (Macauley & Ramadjita, 2015; Rico, 1976). The sorghum varieties of Haiti appear to be relatively poor hosts for the most damageable plant parasitic nematodes.

Phylogeny of the sub-family Telotylenchinae The Dolichodoridae is consider as one of the most diverse nematode family (Geraert, 2011). Three classifications are currently used to approach the systematic of the Dolichodoridae family : Fortuner and Luc (987), Siddiqui (2000) and Geraert (2011). According to the classification of Geraert (2011), the Dolichodoridae have seven subfamilies with around 428 valid species described. The Telotylenchinae, the richest subfamily of the Dolichodoridae family, contains nine genera with 260 valid species. The division of the sub-family of Telotylenchinae into the different genera was based on the morphological variations observed between the specimen, sometimes one single feature was used to differentiate two genera. As example, only the number of lateral lines is worth to differentiate the genera Quinisulcius (5) from Tylenchorhynchus (4) and the abnormal thickened in the tail terminus cuticle is useful to differentiate Paratrophorus (presence) from Tylenchorhynchus (absent). Geraert (2011) affirmed that most of the species distributed over the nine genera were firstly considered as a Tylenchorhynchus species. Based on the Baysian inference trees of the partial 18 SSU rRNA, the D2-D3 28S LSU and the ITS genes, we observed that most of the genera of the sub-family of Telotylenchinae are embedded on each other and have close phylogenetic relationship with Tylenchorhynchus (Fig. 4 & Fig. 16). From the molecular analysis of the recovered populations in Haiti and the sequences of subsequent genera available in GeneBank, we reject the species approach of Handoo et al. (2014b) suggesting monophyly of some species of Bitylenchus from Tylenchorhynchus. We assume that the phylogeny analysis of the sub-family of Telotylenchinae

46 should be approached at genus base otherwise the same genus will be monophyletic and paraphyletic to another genus. Furthermore, the Bitylenchus has been consider as a synonymy of Tylenchorhynchus Geraert (2011) because of few distinctive features between the two genera. Based on the 18S gene, the genera Paratrophorus, Bitylenchus, Neodolichorhynchus, Sauertylenchus, Tylenchorhynchus formed a well supported paraphyletic clade (Fig. 16-A). And, the genera Tylenchorhynchus and Quinisulcius formed a maximally supported monophyletic taxon. Based on the 28S gene, the genera Quinisulcius, Tylenchorhynchus, Bitylenchus and Paratrophorus formed a maximally supported paraphyletic clade (Fig. 16-B). The genera Telotylenchus and Tylenchorhynchus formed a separated paraphyletic clade, maximally supported. The genera Histotylenchus and Tylenchorhynchus formed a separated paraphyletic clade, maximally supported. The genus Trophorus formed a maximally supported monophyletic clade. The baysian inference tree for the ITS confirmed the paraphyletic relationship between Tylenchorhynchus and Quinisulcius also between Paratrophorus and Bitylenchus. The D2-D3 28S gene is relevant for the phylogenetic analysis of the sub-family of Telotylenchinae as Handoo et al. (2014b) have already highlight it . Based on the molecular analysis from this study of the sub-family of Telotylenchinae, we suggest that the genera Paratrophorus, Bitylenchus, Neodolichorhynchus, Sauertylenchus, Histotylenchus, Telotylenchus and Quinisulcius could be combined with Tylenchorhynchus to form a super genus of Tylenchorhynchus sensu lato.

Conclusion The taxonomic list of plant parasitic nematodes associated established in this study showed that the cereals crops of Haiti (Rice, Maize, sorghum) are attacked by a broad range of nematode genera. The prevalence and the density of PPN on cereals in Haiti appear to be strongly influenced by the crops. The density of the known damageable nematodes has a relatively lower density compared to other studies realized in other places of the world. Based on morphometrical characterization of multiple genera, the nematodes of Haiti appear to have a smaller body size compared to the respective description of the same population of these nematode genera worldwide. Finally, further researched should be conducted to assess the pathogenicity of the nematode species recovered on the investigated cereals crops and to study the diversity of nematode associated with some other important crops like: plantain/ banana, potatoes, vegetables, etc.

Acknowledgement All my gratitudes to Prof. Dr. Bert for his unconditional supports. Thank to Prof. Karssen for his help in the identification of the nematodes. Thank to Rolish and everyone in the Nematology Research Unit, UGent.

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