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Insect vectors transmission of phytoplasma to vegetables in Eastern Visayas Philippines

Phytoplasma diseases are found affecting vegetables in Eastern Visayas, Philippines. Identifying the insect vectors that transmit phytoplasma in the field is necessary for the management of this disease. This study was conducted to identify the vectors of phytoplasma affecting bitter gourd, loofah, and string beans. Insects associated with these vegetables that had shown phytoplasma symptoms in the field were collected, mass-reared, and the progenies of mass reared insects were used for transmission to healthy host plants. Phytoplasma detection was done through PCR and nest PCR assays. Phytoplasma was positively transmitted to healthy bitter gourd plants by a cicadellid leafhopper, sp. and brown Hishimonus planthopper, . The ~ 1.25Kb phytoplasma-specific band was Ricania speculum amplified in these insects. The phytoplasmas transmitted by the sp. Hishimonus and produced two different types of symptoms to the bitter gourd, and R. speculum rDNA sequence analysis separated them into two clusters which confirmed that the phytoplasmas belong to two different strains. Although the and Aphis gossypii Aphis craccivora also produced symptoms in bitter gourd and string beans, and the pathogen was detected in the insects, whether they vectored phytoplasma to their respective hosts needs further study, since the symptoms produced were quite different from the ones produced by the cicadellid leafhopper and Ricania speculum .


INTRODUCTION
Phytoplasmas are phloem-limited phytobacteria that lack cell walls and are mainly transmitted by insect vectors in a persistent manner (Weintraub and Beanland 2006). Rojas-Martinez (2009) reported that phytoplasmas are transmitted by phloemfeeding insect vectors most commonly belonging to Homoptera of the families: Cicadellidae, Cixiidae, Psyllidae, Cercopidae, Delphacidae, Derbidae, Menoplidae, and Flatidae. Weintraub and Beanland (2006) listed 92 insect species that transmit phytoplasma diseases, primarily leafhoppers, planthoppers, and psyllids. An additional five confirmed vector species and some previously known vectors with new phytoplasma associations were reported by Weintraub (2007).
The identification of insect vectors that transmit phytoplasma diseases is essential for their management. Beets phytoplasma was reported to be transmitted by the leafhopper, Mirzaie et al 2007). Lettuce phytoplasma is Orosius orientalis ( transmitted by leafhoppers sp. (Borth et al 2006) and Macrosteles Neoaliturus fenestratus (Salehi et al 2006). Khan et al (2003) positively amplified phytoplasma affecting alfalfa from two leafhopper species identified as and Austroagallia avicula Empoasca sp. which indicates that these insects are potential vectors of Alfalfa phytoplasma. Mirzaie et al (2007) reported that transmits the Orosius orientalis garden beet witches' broom. Sugarcane white leaf disease is transmitted by the leafhopper (Hanboonson et al 2006).
Phytoplasma diseases are becoming more common in vegetables in the Eastern Visayas region of the Philippines, specifically in bitter gourd ( ), Momordica charantia loofah ( ), and string beans ( .

Luffa cylindrica
Vigna unguiculata sesquipedalis) Phytoplasmas have also been observed in "Baguio" beans ( ), Phaseolus vulgaris cucumber ( ), and tomatoes ( ) (Borines et al Cucumis sativus Solanum lycopersicum 2020). Typical symptoms include reduced size of new leaves and the plant showing "witches' broom" or bunched apical growth symptoms. Fruit, if produced, is much reduced in size or absent, thereby severely affecting yield.
The insect vectors that lead to the spread of phytoplasma diseases need to be identified for their management, however, knowledge on insect vectors of vegetable phytoplasmas is lacking in the Philippines, particularly for bitter gourd, loofah, and string beans. This insect transmission study aims to identify the insect vectors of the phytoplasmas affecting bitter gourd, loofah, and string bean.

Collection and Mass Rearing of Insects Associated with Phytoplasma Symptoms
Insects associated with phytoplasma disease symptoms (chlorosis and little leaf), namely a cicadellid leafhopper, , and from Ricania speculum Aphis gossypii bitter gourd and loofah, and black aphids from string beans showing little leaf, were collected from vegetable farms in representative areas Baybay City, Ormoc City and Maasin City in the Eastern Visayas region of the Philippines. The brown cicadellid leafhopper, , and were the insects suspected to be vector in R. speculum A. gossypii bitter gourd and loofah phytoplasma, so they were used for insect transmission to bitter gourd. Whereas the black aphid ( ) was the A. craccivora insect suspected to transmit the pathogen to the string bean. The collected insects were placed in mass-rearing cages with healthy bitter gourd plants, which were regularly replaced with new healthy plants. The plants that had been fed upon were regularly checked for phytoplasma disease symptoms, ie, chlorosis and little leaf/ witches broom. The insect species associated with the loofah and bitter gourd phytoplasma symptoms were commonly observed on both plants, so only bitter gourd was used as a healthy host for the transmission studies that involved cicadellid leafhopper, planthopper R. speculum, A. gossypii and green aphid . Mass-reared juvenile insects from the rearing cages where the plants remained healthy were transferred using an aspirator to small plastic containers with field-collected diseased host plants for a 24h acquisition feeding. After the feeding period 10-20 insects were transferred to healthy bitter gourd plants in the greenhouse for insect transmission. Ten healthy plants were used in the transmission study for each insect. As control, the massreared insects from plants which did not show phytoplasma symptoms were transferred directly to healthy host plants. The insects were removed from the cages one week after introduction and destroyed. Throughout the experiment each plant was covered entirely with tulle cloth to avoid escape and unwanted infestation. The test plants were observed for the development of symptoms typical of a phytoplasma disease.

Phytoplasma Detection from Insects
Total DNA extraction, PCR, and nest PCR assays Phytoplasma detection was conducted on insects associated with phytoplasma symptoms of bitter gourd, loofah, and string bean, to confirm whether they harbored the pathogen. Detection through PCR and nested PCR assay was also done on insects that had positively introduced the pathogen to healthy host plants. During the PCR analysis and after the conduct of insect transmission studies, it was observed in one string bean field visit where the plants were showing phytoplasmalike symptoms, a green leafhopper, . was associated with the diseased Empoasca sp plants so samples of these insects were collected and included in the PCR analysis for the presence of the pathogen. However, no transmission study was conducted with this insect since the project was near termination.
amples were -Next day the s centrifuged at 12000rpm for 30mins then washed with 70% ethanol. The pellet was air-dried and re-suspended in 100µL TE (10mMTris-HCl, mM EDTA, pH 8.0) buffer. 1 Two further extractions , first with 100µL phenol:chloroform:isoamyl were performed alcohol (25:24:1) 100µL chloroform. The upper aqueous layer was followed by removed, and 300µL cold absolute ethanol was added to precipitate the nucleic acids. DNAs were centrifuged at 13000rpm for 30mins, washed with cold 300µL 70% ethanol, air-dried, and re-suspended in 10-40µL TE buffer, depending on the size of the DNA pellet. The DNA concentration in each isolation tube was measured through gel electrophoresis together with known concentration of DNA for comparison. Next 2µL RNAse was added to each tube and incubated at 37°C for 30mins. Nucleic acids were stored at 20°C. A concentration of 50-100ng per uL DNA was used in the -PCR analysis. The same protocol was used in extracting DNA from , Aphis craccivora the string beans aphid.
Initial PCR analysis was done using the universal primer P /P for Phytoplasma 1 7 developed by Deng and Hiruki (1991), followed by a nested PCR using the primer R F n/R R (Gundersen and Lee 1996, Lee et al 1995 conditions were as follows, initial denaturation at 94ºC for 2mins, 30 cycles of denaturation at 92ºC for 1min, annealing at 55ºC for 15s, and extension 72ºC for 30s, and a final extension at 72ºC for 10mins. The PCR products were subjected to agarose gel electrophoresis using 1% agarose gel, stained with Gel Red Stain® (Biotium). The bands were viewed under a UV trans-illuminator attached to an Alpha Digi-Doc Documentation System. The insects that produced positive transmission of phytoplasma to healthy bitter gourd and loofah and showed positive phytoplasma bands in the PCR analysis were considered as the phytoplasma vector of these host plants.
16SrDNA Sequence Analysis

Insect Transmission
All the bitter gourd test plants exposed to the three phytoplasma infected insects (brown cicadellid leafhopper, and ) exhibited the phytoplasma-R. speculum, A. gosypii like symptoms (Figure 1). However, there were slight differences in the symptoms l produced by these insects (Table 1). String bean aphid, A. craccivora also produced the little leaf symptom in its host plant. The inoculated plants were also subjected to PCR and nest PCR which showed positive results for phytoplasma. The cicadellid leafhopper with light brown and dark brown markings on the wings transmitted the phytoplasma that produced the typical little leaf with witches' broom symptoms in bitter gourd. Affected plants produce very tiny pale green leaves, witches' broom and shoestring symptoms, and generalized stunting (Figure 1a), but no foliar chlorosis was evident. The cicadellid leafhopper was small, approximately 5mm for the adult (Figure 1b) and 2mm for the nymph (Figure 1c). Figure d is a 1 close-up photo of the insect viewed under a USB microscope. It was morphologically identified as one of the species and closely resembles Hishimonus Hishimonus phycitis Distant, a cicadellid leafhopper associated with citrus described by Du and Dai (2019).
spp. are described by Du and Dai (2019) as small species, Hishimonus 3.0-5.0mm in length. Coloration usually greenish or yellow with legs stramineous, usually with symmetrical brown markings dorsally on the head, pronotum, and forewing, forewings silvery-white with dark brown mottling, and usually with a large brown semicircular spot at mid-length of commissural margin forming, when the wings at rest, a conspicuous median diamond-shaped spot with that of the opposite wing.
The leaves of plants introduced with the brown planthopper, (Figure R. speculum 2a) were also reduced in size (but not very tiny) and did not exhibit the typical witches broom symptoms but showed foliage chlorosis. No thickening of the leaves was observed (Figure 2b). On the other hand, the bitter gourd plants introduced with the green aphid, produced tiny leaves and chlorosis. Unlike the chlorosis A. gossypii, exhibited by plants introduced with , the leaves were thickened ( Figure  R. speculum 3). The thickening of the leaves with yellowing is also a characteristic symptom of "Namamarako" disease caused by the cucurbit aphid-borne yellow virus (CABYV). The namamarako-infected leaves, however, although thickened usually do not turn very tiny as was observed in the plants under this study. Yellowing and tiny leaf size are definitely symptoms of phytoplasma and thickening of the leaves may also be due to phytoplasma infection. The juvenile progenies of mass-reared field-collected aphids used in the transmission studies were assumed free from any pathogen. If phytoplasma did not cause the thickening of the leaves, it could also have been due to the direct effect of aphid feeding. Nang et al (2014) positively detected phytoplasma in bitter gourd little leaf in Myanmar that showed small, yellowish green, thickened and puckered leaves, and the internodes were also thickened. Since reports on insect vectors of phytoplasmas to host plants were mostly identified as leafhoppers, planthoppers and psyllids (Rojas-Martinez 2009, Weintraub andBeanland 2006, Weintraub 2007), even if the pathogen was detected in the aphids, we could not conclude that this insect is really a vector of phytoplasma. Cainelli et al (2007) had detected the pathogen in aphids but, according to their research, the aphids did not transmit phytoplasma to apple plants.
On the transmission study on string beans using results showed A. craccivora, that the test plants introduced with also showed little leaf symptoms A. craccivora ( Figure 4). As with bitter gourd phytoplasma, there was also the need to confirm the phytoplasma transmission to string beans by . A. craccivora

Detection of Phytoplasma from Arthropod Vectors
Phytoplasma detection from the suspected insect vectors through PCR and nest PCR assays were conducted using the universal primer P /P and to 1 7 R F n/R R 16 2 16 2 confirm that these insects harbored phytoplasma in their bodies. A phytoplasmaspecific band of ~1.25Kb was positively amplified from the leafhopper Hishimonus sp. that had positively transmitted the pathogen to the bitter gourd, causing the typical witches broom symptoms in healthy plants in the screen house ( Figure 5). No band was initially detected from field-collected (Rn) DNA in the initial R. speculum PCR run. The phytoplasma-specific band was also amplified from DNAs A. gossypii (AGA and AGP), which confirmed that aphids carry the pathogen in their bodies. Phytoplasma bands were also amplified from infected host plant DNAs such as loofah (PH) and bitter gourd (AG, AV1, and AV2) DNAs.
The nest PCR assay was repeated with the DNA sample (Rn), R. speculum cicadellid leafhopper (CLH) together with the string bean aphid (SBA), and green leafhopper sp. (Esp) DNA since it was also reported to transmit loofah Empoasca phytoplasma to plants (Khan et al 2003) and this insect was observed in a string bean field that showed phytoplasma-like infections. Figure 5. Nest PCR amplification results from insects used in transmission study. green aphids from bitter gourd (AGA) and loofah (AGP), (Rn) and Aphis gossypii Ricania speculum cicadellid leafhopper sp. (CLH). Positive phytoplasma band amplifications were Hishinomus obtained from the green aphid (AGA). Amplifications were also obtained from field-infected loofah (PH) and bitter gourd (AG, AV1, and AV2) that served as controls The PCR products were subjected to gel electrophoresis together with amplified phytoplasma from infected plant DNAs of loofah little leaf (Pi), Baguio bean little leaf (BB), bitter gourd little leaf (AL2), and healthy bitter gourd DNA (C) (Figure 6). The ~1.25Kb band was amplified from the cicadellid leafhopper and R. speculum, which transmitted the disease to the bitter gourd in the screen house. The same band was also detected from string bean aphid (SBA), which transmitted the disease to string beans. The cicadellid leafhopper (CLH) and (Rsp) R. speculum phytoplasma DNA was again amplified in another PCR run (Figure 7). No band was detected from sp in two PCR analys s. Empoasca either of the e The R F n/R R amplified products from insect vectors were subjected to gene 16 2 16 2 sequencing . However, the phytoplasma amplicon from diverged slightly from R. speculum most loofah phytoplasma genes and other controls (92-95% similar), and even more for the string bean aphid, which only shares 87-90% similarity to the controls. These results suggest that the strains of phytoplasma transmitted by R. speculum to bitter gourd and loofah in the field transmitted by the are different from those cicadellid leafhopper sp. based on the symptoms they produced in the Hishimonus bitter gourd host in this study. It corroborates well with the symptoms noted on the introduced plants, which varied between sp. that caused the very tiny Hishimonus leaves causing the typical witches broom symptoms, and transmitted R. speculumphytoplasma just reduced the affected leaf's size a little together with leaf yellowing symptoms. The strains from string bean aphid are just 87-90% similar A. craccivora to the controls used, suggesting another strain of phytoplasma carried by the string bean aphid. Figure 10 shows the multiple sequence alignment of phytoplasma amplicons from host plants, vectors, and controls showing a high consensus value of 90% from the 260 to 326 genomic nucleotide position indicated by the red-colored nucleotides. Few single nucleotide polymorphisms (SNPs) and indels were observed, with S1R16 (string bean phytoplasma) showing the most frequent variation other than the outgroup reference Rice. Figure 11, on the other hand, shows the phylogenetic dendrogram of phytoplasma nucleotide sequences from the insect vectors (Rn, CLH, LC2, and SBA), together with plants ( , and controls analyzed using UPGMA method. The data show that the strains were grouped into two main clusters. A tight cluster comprised of cicadellid leafhoppers ( Hishimonus sp.) (CLHR16, LC2R16), string bean aphid ( ) (SBAR16), bitter gourd, A. craccivora and loofah phytoplasmas (ALR16, AV2R16, PHgR16, PIR16, and PVR16) was noted, which also represents the most recent type of strains. By contrast, the other group representing early lineages of Phytoplasma strains, including tomato stunt phytoplasma TG R16, Rn R16 and Baguio bean phytoplasma BB R16, R. speculum showed expansion probably by frequent nucleotide substitution events.
These overall results implied that cicadellid leafhopper, sp., and Hishimonus brown planthopper, are confirmed vectors of two strains of R. speculum phytoplasma based on their positive transmission to healthy bitter gourd hosts, positive detection through PCR and Nest-PCR, and rDNA sequencing and phylogenetic analysis. Athough the green aphid, and black aphid A. gossypii, transmission to bitter gourd and string bean also produced the phytoplasma-like little leaf symptoms and the pathogen was detected in the insect DNAs, further studies should be done to confirm their positive transmission of phytoplasma to host plants. Cainelli 2007 reported that aphids seem to acquire quite a high load of the apple phytoplasma but the concentration of the pathogen in these insects was lower when compared to the concentrations detected in psyllids which transmitted the disease to apples. They proposed that aphids don't play any relevant role in apple phytoplasma spreading. Figure 10. Alignment of the 19 nucleotide sequences of Phytoplasma-specific amplicons from host plants, insect vectors, and controls. (Multiple sequence alignment using MultAlin). Note: TG represents Phytoplasma rDNA from tomato, AG is Phytoplasma amplicon from , A. gossypii AL, and Av2, -Phytoplasma amplicon from infected Bitter gourd, CLH from cicadellid leafhopper ( sp.), PH, PI, and PV-Phytoplasma from infected sponge gourd, LC2-Hishimonus from cicadellid leafhopper sp. from the screen house, Rn-from , SBA Hishimonus R. speculum from string bean aphid and S1 from infected string bean. The others are controls from the genebank Figure 11. Phylogenetic dendrogram of nineteen phytoplasma nucleotide sequences from host plants (tomato-TG, Baguio bean-BB, bitter gourd -AG, AL, and AV2, loofah -PHg, PI, PV), insect vectors (Rn, CLH, LC2 and SBA) and the rest are controls

CONCLUSION, IMPLICATIONS AND RECOMMENDATIONS
Phytoplasma was positively transmitted to healthy bitter gourd plants by a cicadellied leafhopper, . and brown planthopper The Hishimonus sp R. speculum. Hishimonus sp.
R. produced the typical little leaf/witches broom symptoms, while speculum transmitted the yellows-type phytoplasma disease symptoms. Phytoplasma was positively detected in these insects through PCR and nest-PCR assay. These results confirmed that these insects are vectors of two different strains of phytoplasma to bitter gourd. Although the green aphid, transmission A. gossypii produced the little leaf and chlorosis symptoms, and the pathogen was positively detected in the insect, whether or not the green aphid vectored phytoplasma to the test plants needs further study since the symptoms produced were quite different from that of the symptoms produced by the cicadellid leafhopper ( sp.) Hishimonus and only one literature was encountered which reported that aphids are able to transmit phytoplasma. The same is true for further study is needed to A. craccivora, prove whether it transmits the little leaf phytoplasma to string bean. Further study also needs to be conducted on the possibility of phytoplasma transmission by Empoasca sp. to string bean, bitter gourd and loofah, or whether it transmits a viral pathogen instead.