Introduction

Papaya is one of the important horticultural crops in Indonesia. Since 2017, the papaya production in Indonesia has increased rapidly, from 875.112 tons in 2017 to 1.168.266 tons in 2021. However, several viruses have been reported to attack papaya plants, including the Potyvirus group, such as Papaya ring spot virus strain P (PRSV-P) [1] and the Begomovirus group, including Papaya leaf curl virus (PalCV), Papaya leaf crumple virus (PalCrV), Chilli leaf curl virus (ChilCuV), Tomato leaf curl New Delhi virus (ToLCuNDV), and Croton yellow vein mosaic virus (CYVMV) [2]. The first reported virus attack on papaya in Indonesia was Papaya ring spot virus strain P (PRSV-P), which was found in Nanggroe Aceh Darussalam Province [3]. Today, PRSV attacks have spread to almost all provinces in Sumatra and Java [4].

After reports of PRSV in Indonesia, there were reported the presence of Begomovirus group that attacks papaya plants in Bengkulu Province [5]. The symptoms of papaya plants attacked by Begomovirus are yellowing, malformations of the leaves, and sometimes brown spots. This report indicates that the Begomovirus has entered Indonesia. Begomovirus is transmitted by whitefly (Bemisia tabaci) [6]. Lampung and Bengkulu are geographically very close, so it is necessary to detect the presence of this virus in papaya plants in Lampung Province.

Materials and methods

To identify the Begomovirus in papaya, DNA was extracted from seven samples representing different papaya growing areas using a Plant Genomic DNA Mini Kit (Geneaid Biotech Ltd., Taiwan), following the manufacturer’s instructions. Genomic DNA from the samples was subjected to PCR using universal primers for Begomovirus which amplify parts of AC2 and AC1, as well as SPG1 (5′CCCCKGTGCGWRAATCCAT-3′) and SPG2 (5′ATCCVAAYWTYCAGGGAGCTAA-3′) which amplify nucleotides encoding Begomovirus transcriptional activator protein (TrAp) and replication-associated protein (Rep) genes [7]. PCR was performed with an initial denaturation cycle of 95 °C for 1 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 50 °C for 15 s, extension at 72 °C for 10 s, and final extension at 72 °C for 5 min. The PCR products were then checked by using electrophoresis in 1% agarose gel. The PCR products were then sequenced using the dideoxy (Sanger) approach.

The obtained sequence then compared to the gene bank using BLAST software available on the National Center For Biotechnology Information (NCBI) website, www.ncbi.nlm.nih.gov. This analysis was used to determine the degree of kinship between our isolates and the Begomovirus strains that have been registered in the GenBank database (NCBI). Furthermore, several strains were selected with a similarity percentage close to 100%. Multiple sequence alignment and phylogenetic tree construction were analyzed using the MEGA11 program. The phylogenetic tree was arranged using the UPGMA algorithm, with grouping stability using bootstrap analysis with 1000 replications.

Samples that have been detected using PCR are then subjected to pathogenicity tests using three types of plants to confirm the symptoms that arise. Incoculum plants and healthy plants were placed in a glass cover which had been infested with Bemisia tabaci imago. A positive result of the pathogenicity test is indicated by the appearance of symptoms on healthy plants that are the same as those on inoculum plants.

Result and discussion

Observations in areas of papaya plantations in Pesawaran, Lampung Province showed that in seven sampling points the yellowing disease of papaya was detected. Among these, there were seven samples of papaya plants infected with Begomovirus,

Infections that occur in papaya plants will cause several symptoms that are expressed through plant parts such as leaves and other plant parts. Papaya leaves show symptoms of mosaic, leaf curl, yellowing, and malformations in papaya plants (Fig. 1). In gardens with more severe symptoms in papaya plants are the leaves that fall cause the papaya plants to die over time (Fig. 2). In addition, several Bemisia tabaci vector insects were found around symptomatic papaya plants. The results of these observations indicated that there was a spread of yellow virus (Begomovirus) in papaya plants in Lampung Province.

Fig. 1
figure 1

Symptoms on papaya leaves infected with Begomovirus; Mottle A Yellowing B and C Mosaic D and shoe string E

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Fig. 2
figure 2

Comparison of papaya fields on Begomovirus attack with low A and high B attack rates

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Observations of papaya fields in Pesawaran, Lampung Province showed that Begomovirus attacks had different intensities. Papaya fields in Pesawaran have an incidence of around 50–100%, while the severity of disease in the field ranges from 20 to 95%. This phenomenon showed that the plant could produce fruits while infected by this virus, although in a small yield (Fig. 3).

Detection based on viral nucleic acid in each category of papaya plants in Pesawaran using universal primers SPG1/SPG2 showed positive results for Begomovirus infection with visible DNA bands measuring ± 900 bp (Fig. 4).

Fig. 3
figure 3

Results of DNA amplification using SPG1/SPG2 primers on papaya leaf samples from several districts/cities in Lampung Province. Markers (M); sample 1 (1); sample 2 (2); sample 3 (3); sample 4 (4); sample 5 (5); sample (6), and sample 7 (7)

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Fig. 4
figure 4

Phylogenetic tree constructed using UPGMA Algorithm from AC2 and AC1 Gene

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Ageratum yellow vein virus (AYVV) was found in the papaya samples through direct nucleotide sequencing with SPG1/SPG2 primers for the AC2 and AC1 genes of Begomovirus. This was confirmed by the BLAST analysis of 97.52%.The phylogenetic results show that AYVV from papaya samples are closely related to AYVV groups from several other countries with 98% homology (Fig. 4). There are 23 base pairs that are the same out of 235 base pairs. Phylogenetic analysis also showed that the AYVV of tomato plant samples in Lampung Province were in the same cluster as AYVV from Japan (KC677733), Indonesia (AB100305), Nepal (KC282641), Taiwan (DQ866134), Thailand (JN809811), and Vietnam (MW012407). Interestingly, most of the AYVV found in several countries are AYVV that attack Ageratum and several types of Solanaceae plants such as tomatoes, eggplants, and chilies. Meanwhile, what is found in Indonesia is AYVV which attacks papaya plants with a disease incidence rate of almost 100%. Thus, this finding poses a serious threat to papaya cultivation in Indonesia. Not only threatening Indonesia, this finding can also threaten papaya- producing countries such as India, Brazil, China, Mexico, and several other countries [8]. The main host of AYVV is Ageratum, a cosmopolitan weed that is often found around cultivated fields and is mostly attacked by jaundice caused by Begomovirus. This suggests that Ageratum acts as an alternative host and reservoir for Begomovirus.

A pathogenicity test was carried out to confirm that the symptoms present in the inoculum plants were the same as those inoculated with Begomovirus using the Bemisia tabaci vector. Pathogenicity test results on three types of plants showed that the symptoms of the inoculums occurred in the inoculated plants. Symptoms that appear can be seen in chili leaves that curl (Fig. 5A), papaya leaves with malformations (leaves do not grow properly), curly leaves (Fig. 5B), and eggplant leaves with chlorosis yellow spots (Fig. 5C), in severe infections causing stunted plants. There are five plants that are used to confirm symptoms. in the three types of plants, five out of five plants showed symptoms of disease (100% infected).Further tests were not carried out due to research limitations.

Fig. 5
figure 5

Symptoms in plants inoculated with Begomovirus; on chili plants A on papaya plants B on eggplant C

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Begomovirus infection in plants will generally cause symptoms such as leaf curling, yellowing, mosaicism, and stunting [9].

Disease epidemics are influenced by the active role of insect vectors, the adequacy of inoculum sources in the field, the use of susceptible varieties, and environmental factors suitable for the development of insect vectors and pathogens. Based on the research that has been done, information about the spread of Begomovirus in various plants is needed, considering that this virus easily moves between plants from various families. Therefore, accurate detection using molecular techniques is one way to obtain information on the spread of the virus, so that control strategies can be applied to minimize the spread of the virus in the field.