ZYMV is becoming a serious pathogen in most cucurbit growing regions of the world where the infection rates of at least 40% has been reported in tropical and sub-tropical regions. Disease surveys in pumpkin have indicated ZYMV disease incidence levels reported up to 75 % in dry season in Trinidad .
Natural populations of RNA viruses rapidly generate genetic diversity because of a combination of high mutation rates, rapid replication, recombination events, high frequency of occurrence and a variety of strains . In this study, phylogenetic analysis revealed that Trinidad isolates form a new genotype ‘ZYMV-Trini’ since they have variations of 5.9-6.0 % in complete nt sequences as compared to their closest known relatives, including isolates NAT and AG (Israel) and SE04T (Slovakia). The within-virus species genotype classification system adopted a neutral nomenclature involving letters of the alphabet and Latinized numerals that avoid potentially misleading names [29, 30]. Phylograms of aa sequences also showed the ZYMV-Trini isolates forming a separate cluster for HC-Pro, CI, and NIb genes in this study. In case of the phylogram of the P1 gene sequences, the isolate TW-TN3 (Taiwan) was closely related to the Trini isolates, with 2.3–2.4 % nt variation level.
The capsid protein (CP) gene of potyviruses is widely used as a valid typing tool to differentiate among isolates . However, comparison of complete genome sequences allows for a more complex analysis of virus variability and may provide information on the evolutionary history and existence of major evolutionary events, such as recombination, as was demonstrated for various potyviruses [14, 41]. Among the gene sequences in the polyprotein of ZYMV-Trini isolates, NIb and CP are highly conserved but the HC-Pro gene had the maximum variation in aa sequences, as compared to the closely related isolates. In a phylogram constructed based on coat protein aa sequences, all previously reported ZYMV-coat protein sequences from Trinidad and Tobago and ZYMV-Trini isolates from this study get grouped together and this may suggest that all the ZYMV isolates from Trinidad and Tobago belongs to the same genotype (data not given). Among the other reference isolates, ZYMP13PREP from Reunion Island had maximum variations from the Trini isolates, viz., 23.6 % with complete nt genome, and 18.6 % with CI and 7.1 % with NIb aa sequences (Table 1).
Aphids are the most successful vectors of potyviruses, due to an array of generic and specific features they possess , including precision delivery of viral particles via the stylet, parthenogenetic mode of reproduction within a short span of time, diverse range of host plants, survival in adverse conditions and the ability to disseminate over long distances [28, 33]. Katis et al.  reported the most abundant aphid vectors of ZYMV in a study in Greece included M. persicae, Aphis gossypii and Aphis spiraecola. We also reported A. gossypii as a vector of ZYMV in Trinidad in our earlier preliminary study  and it was reconfirmed in the current study. The relative standard curve method through RT-qPCR effectively detected the incremental increase of ZYMV-RNA targets in pumpkin seedlings following transmission through A. gossypii. Vector transmission occurs as a result of interaction between the aphid stylet, and viral proteins of ZYMV such as coat protein (CP) and helper component proteinase (HC-Pro). Specifically, the DAG motif on the CP interacts with the PTK region of the HC-Pro, and a secondary motif on the HC-Pro (KLSC) interacts with the aphid stylet . Volunteer cucurbitaceous crop and weed plants also act as infection sources for ZYMV spread to cucurbit crops [5, 6, 23].
Generally, ~ 20% of viral plant pathogens are known to be seed-transmitted. Seed to seedling transmission rate was earlier reported for ZYMV at a low level (1.6%) [17, 37]. A similar trend was also observed in this study, with only 2% seed transmission rate. The vertical transmission of ZYMV by seed is less common than horizontal transmission via aphids and also many studies support the hypothesis that the insect vector is an important factor in inducing virus variation . In both aphid and seed transmission experiments, ZYMV increments in the plant were steady up to 37 days but a rapid surge was observed by RT-qPCR analysis between 37 and 57 days in this study.
Through recombination, viruses gain pathogenicity or virulence, and the ability to invade new hosts [13, 16]. Recombination is advantageous for RNA viruses as it can create high fitness genotypes more rapidly than mutation alone . This study also supports the hypothesis that recombination is a dominant feature of ZYMV evolution as in other RNA viruses. Recombination sites detected in silico using RDP5 software suggested that the entire ZYMV genome is prone to recombination, though hotspots are concentrated in P1, HC-Pro, P3 and CP gene sequences in several isolates. Recombination breakpoints in the ZYMV-Trini isolates were noticed in P1 and between P3 and NIb gene sequences. Maina et al.  earlier reported the same pattern of recombination breakpoints in ZYMV populations from East Timor and northern Australian cucurbit crops. Natural recombinants may emerge in virus populations only if they maintain relatively good fitness that includes preserving the functionality of each viral protein and the functional interactions between proteins [25, 31]. For plant viruses, the recombination rate might be much higher than expected, whereas rates for potyviruses may be up to ~ 25 % although only a small fraction of the generated variants emerge in the population due to strong selection pressure .
Isolates SE04T (Slovakia), AG (Israel) and NAT (Israel) were determined to be major parental contributors, circumstantially, for the genome architecture of ZYMV-Trini isolates. These parental isolates have 98.0-98.4 % nt identities among themselves and 93.4–94.1 % similarities with ZYMV-Trini isolates. Cucurbit cultivation in the Caribbean islands including Trinidad and Tobago is mainly dependent on imported seeds from different countries, and seed producing countries such as Israel, and China play a significant role in seed transfer. ZYMV can also move to new locations in ZYMV-infected fruit from which aphids can acquire and spread the virus . Introductions could also occur from migrating birds carrying virus-infected seed in their intestines or discarded infected cucurbit fruit left behind by fishermen from neighbouring countries camping on the shore . Maina et al.  reported the absence of genetic connectivity between ZYMV sequences from Papua New Guinea (PNG) and those from Australian or East Timor. The highest nucleotide similarity between a ZYMV sequence from PNG and elsewhere was 92.8% and the authors suggested the divergence could be due to a single introduction of ZYMV into PNG with subsequent evolution to adapt in this new environment.
It is also interesting to know that ZYMV-Trini isolates may have contributed as a major parent for isolates such as Z5-1 (Japan), Z-104 (Italy) and ZYMV-WS (China) for various genome fragments. However, more data need to be generated from the Caribbean island countries to study the genome dynamics of ZYMV-Trini isolates and their genetic connectivity among the isolates from neighbouring countries.
This study provides the first report of the complete nucleotide sequence of ZYMV from Trinidad and Tobago and further also highlights that recombination is a major driving force in the evolution and emergence of new variants of ZYMV. It is also noteworthy to understand the complexity of the variability of ZYMV isolates in order to derive effective field control measures.