Research Note
Detection of ‘Candidatus Phytoplasma luffae’ in Sponge Gourd, Bitter Gourd, and Bamboo from Laguna in Luzon, Philippines
Filomena C. Sta Cruz
1,*, Nuredin Habili
2, Qi Wu
2, Rezel B. Sagarino
3and John W. Randles
41Institute of Weed Science, Entomology and Plant Pathology, College of Agriculture and Food Science, University of the Philippines Los Baños, College, Laguna 4031, Philippines
2The Australian Wine Research Institute, Corner of Hartley Grove and Paratoo Road Urrbrae SA 5064, Australian
3Graduate School, University of the Philippines Los Baños, College, Laguna, Philippines
4Waite Diagnostics, School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus Urrbrae SA 5064, Australian
*Author for correspondence; Email: [email protected]
Received: 11 March 2021/ Revised: 10 May 2021/ Accepted: 26 May 2021
In the Philippines, phytoplasma disease symptoms of little leaf and witches’ broom have been observed in sponge gourd (Luffa sp.), bitter gourd (Momordica charantia) and bamboo (Bambusa vulgaris). However, the cause of the disease remained unidentified. Molecular analysis was conducted to detect the presence and identify the phytoplasma associated with the diseased plants. Nested PCR has detected the presence of phytoplasmas in sponge gourd, bitter gourd and bamboo showing symptoms of little leaf and witches’
broom. Nucleotide BLAST and phylogenetic analyses of the 16S rRNA gene have identified ‘Candidatus Phytoplasma luffae’ belonging to group 16SrVIII in all three crops with highest similarity (98.04%, 99.51%, and 99.25%) to the loofah phytoplasma reference strain from Taiwan. This is the first report of detecting the same species of ‘Ca. Phytoplasma’ in three different genera of plants from Laguna located in Luzon island, the Philippines.
Keywords: phytoplasma 16S rRNA gene, ‘Candidatus Phytoplasma luffae’ 16SrVIII group
Abbreviations: PCR – polymerase chain reaction, BLAST –basic local alignment sequence tool
INTRODUCTION
Phytoplasmas (‘Candidatus Phytoplasma’) are obligate prokaryotic wall-less plant pathogenic bacteria, which are mainly transmitted in plants by the phloem-feeding insects such as leafhoppers and planthoppers and through vegetative propagation. They cause diseases in many economically important vegetables, pulse, oil crops, cereals, sugar crops, fruit crops, ornamentals, medicinal plants, palm species, forest trees and weeds (Bertaccini et al. 2014; Bertaccini et al. 2018; Rao et al. 2018). The cucurbits are among the vegetable crops that are commonly affected by phytoplasmas (Kumari et al. 2019).
Symptoms of phytoplasma disease in vegetables include phyllody, flower virescence, big buds, little leaf, and witches’ broom.
In the Philippines, the rice orange leaf is the first known phytoplasma disease, which has been reported in the late 1980’s (Hibino et al. 1987). Later, diseases with phytoplasma symptoms of little leaf and witches’ broom have been observed in sponge gourd, bitter gourd, and bamboo grown in Los Baños, Laguna. However, the cause of the disease in these crops remained unidentified. It is important to determine if phytoplasma is the causal agent, so that appropriate measures can be taken to prevent the spread, and manage the disease.
In other countries, little leaf and witches’ broom diseases affecting sponge gourd or loofah and bitter gourd are associated with ‘Candidatus Phytoplasma’
belonging to three different 16S ribosomal RNA groups.
In Taiwan, the phytoplasma associated with loofah witches’ broom is ‘Ca P. luffae’ classified under the group June 2021
16SrVIII, subgroup A (16SrVIII-A) of which two strains, namely LfWB (GenBank accession no. AF248956) and LfWB#2 (GenBank accession no. AF353090) have been identified (Bertaccini et al. 2014; Davis et al. 2017).
Phytoplasmas from other ribosomal groups such as the
‘Ca. P. pruni’ group 16SrIII in Brazil (Montano et al. 2007), and ‘Ca. P. asteris’ group 16SrI in India are associated with 'loofah witches’ broom disease (Kumar et al. 2010).
Phytoplasmas associated with bitter gourd are ‘Ca. P.
pruni’ and ‘Ca. P. asteris’ (Montano et al. 2000; Win et al.
2014; Venkataravanappa et al. 2017). Phytoplasma ‘Ca. P.
asteris’ has also been detected infecting bamboo in Korea (Jung et al. 2006).
This study was conducted to determine the presence and identify the phytoplasma associated with little leaf and witches’ broom disease of sponge gourd, bitter gourd and bamboo grown in Laguna, Philippines.
MATERIALS AND METHODS
In this study, molecular analysis was conducted at the Waite Diagnostics, School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus Urrbrae in Australia, to determine the presence and identify the phytoplasma in the symptomatic sponge gourd, bitter gourd, and bamboo collected in 2014 from Los Baños, Laguna. Plants were showing phytoplasma disease symptoms of little leaf and proliferation of axillary buds resulting in witches' broom growth (Fig. 1). For each crop, leaf samples from four symptomatic and four healthy plants were collected. The total nucleic acid was extracted from 0.2 g leaf tissue using the method described by Mackenzie et al. (1997) except that, instead of columns, a a suspension of silica (Sigma-Aldrich Cat # S-5631) was used to absorb the nucleic acids (Habili et al. 2012). This
was followed by two washes in 4 M guanidine and 75%
ethanol, respectively. Then total nucleic acids were eluted in 0.2 ml 0.01 M Tri-HCl at pH 8.5.
Presence of phytoplasma was tested by PCR using the universal primer pair P1/P7 followed by nested primer pair R16F2 (5’ACG ACT GCT GCT AAG ACT GG 3’) and R16R2 (5’TGA CGG GCG GTG TGT ACA AAC CCC G3’) that amplifies the phytoplasma 16S rRNA gene (Lee et al.
1993). Each reaction of 10 μL consisted of 1 μL nucleic acid extract (50 ng) and 9 μL of PCR master mix from Promega (5X Green GoTaq Ref 7911 Madison, Wisconsin) according to the manufacturer’s protocol. The reaction was subjected to the following amplification conditions:
denaturation of one cycle at 96°C for 4 min, followed by 35 cycles of 94°C for 40 sec, annealing at 54°C for 40 sec, extension at 72°C for 1 min, and a final extension at 72°C for 7 min. Healthy plant DNA samples and water were used as negative controls. Amplicon from an infected plant of each crop was cloned into the plasmid vector pCR2.1-TOPO (ThermoFisher), according to the manufacturer’s protocol. Recombinants were identified by blue-white colony screening and restriction enzyme digestion. The recombinant plasmid DNA was extracted using the Qiagen plasmid Miniprep kit and sent to Bioneer (South Korea) for Sanger sequencing using either the M13 forward/reverse primers or the R16F2 and R16R2.
Sequence alignment was performed using BLASTn and phylogenetic analysis of the isolates was conducted using the MEGA7 software (Kumar et al. 2016). The presence of phytoplasma in the diseased plants with little leaf and witches’ broom symptoms was further confirmed by PCR test using another set of sponge gourd and bitter gourd samples collected in 2017 from Los Baños and Pila, Laguna.
Fig. 1. Phytoplasma disease in a) sponge gourd; b) bitter gourd; and c) bamboo plants showing little leaf and witches’
broom symptoms.
RESULTS AND DISCUSSION
The expected PCR product of about 1.2 kbp was obtained from four infected samples each of bitter gourd, sponge gourd and bamboo. No corresponding amplicons were obtained in healthy and blank samples. Nucleotide BLAST search revealed that phytoplasmas detected in clones of sponge gourd, SG_LB14-Laguna (1225 bp), bitter gourd, BG_LB14-Laguna (1019 bp) and bamboo, BB_LB14 -Laguna (1193 bp) belong to ‘Ca. P. luffae’. Based on the pattern derived from the query of 16S rDNA F2nR2 fragment, all shared highest sequence identity with the reference 16Sr group VIII, subgroup A, ‘Ca. P. luffae’
LfWB and LfWB#2 strains from Taiwan. Sponge gourd, SG_LB14-Laguna (Genbank Accession No. MW074923), bitter gourd BG_LB14-Laguna (Genbank Accession No.
MW074922) and bamboo BB_LB14-Laguna (Genbank Accession No. MW074924) have sequence identities of 98.04%, 99.51%, and 99.25%, respectively with the two
‘Ca. P. luffae’ reference strains (Table 1). Furthermore, they have a sequence identity of 97.96, 99.41, and 99.16% to
‘Ca. P. luffae’ (NCBI Genbank Accession No. AF086621.2) from Taiwan (Ho et al. 2001), and also from bitter gourd in Taiwan (Genbank Accession No. AB667970) (Bau et al.
unpublished).
Following the rules for description of ‘Ca. Phytoplasma’
suggested by the IRCPM Phytoplasma/Spiroplasma Working Team-Phytoplasma Taxonomic Working Group (IRPCM 2004), all strains in this study that have sequence identities (98.04 - 99.51%) greater than the 97.5%
threshold value, belong to the previously described ‘Ca. P.
luffae’ strain, 16SrVIII group. Identity of the phytoplasma and the grouping to which they belong was further confirmed by phylogenetic analysis. The phytoplasma detected in the three crops are all clustered to the same clade of the ‘Ca. P. luffae’ 16Sr group VIII but not with either ‘Ca. P. pruni’ group 16SrIII or ‘Ca. P. asteris’ group 16SrI (Fig. 2). PCR test using another set of bitter gourd and sponge gourd samples collected in 2017 from Los Baños and Pila, Laguna has confirmed the presence of phytoplasmas in diseased plants with little leaf and witches’ broom symptoms. In addition, infected plants Table 1. Sequence identities obtained by nucleotide BLAST search for the phytoplasma 16S rRNA gene from sponge gourd, bitter gourd and bamboo grown in Laguna, Philippines.
Phytoplasma Isolates GenBank
Accession No. Host Country
Nucleotide Sequence Identity (%) Sponge gourd
SG_LB14-Laguna (MW074923)
Bitter gourd BG_LB14-Laguna
(MW074922)
Bamboo BB_LB14-Laguna
(MW074924)
‘Ca. P. luffae’ LfWB AF353090.1 Loofah Taiwan 98.04 99.51 99.25
‘Ca. P. luffae’ LfWB#2 AF248956.1 Loofah Taiwan 98.04 99.51 99.25
‘Ca. P. luffae’ AF086621.2 Loofah Taiwan 97.96 99.41 99.16
‘Ca. P. luffae’ AB667970.1 Bitter gourd Taiwan 97.96 99.41 99.16
Fig. 2. Phylogenetic tree based on the 16S rRNA gene sequence showing the three ‘Candidatus Phytoplasma’ isolates (shown in arrows) from sponge gourd (SG_LB14-Laguna, bitter gourd (BG_LB14-Laguna) and bamboo (BB_LB14-Laguna) aligned with representatives of the ‘Ca. P.’ groups infecting cucumber, peach, chayote and soybean. Sequences were aligned by ClustalW with default settings and Tree was constructed using the Neighbor-joining Method by MEGA7 software, and Bacillus subtilis was used as an outgroup. Genbank accession numbers are specified in the tree. Numbers on branches are bootstrap values obtained from 1,000 replicates. Only bootstrap values higher than 50 are shown.
also showed symptoms of phyllody. Phytoplasma was detected in 10 out of 10 sponge gourd and 10/10 bitter gourd samples from Los Baños, and 10/10 bitter gourd samples from Pila. The expected product of about 1.2 kbp was amplified by nested PCR as shown by representative samples in Fig. 3.
Recently, Borines et al. (2020) has detected the presence of phytoplasma in sponge gourd and bitter gourd collected in 2015 from Eastern Visayas, Philippines.
In this study, we report the presence of phytoplasmas belonging to ‘Ca. P. luffae’ 16SrVIII group in sponge gourd and bitter gourd with little leaf and witches’ broom disease from Laguna province in Luzon. These two studies showed that little leaf and witches’ broom disease of sponge gourd and bitter gourd found in two (Luzon and Visayas) of the three major islands in the country are caused by the same phytoplasma loofah strain. In addition, bamboo was identified in our study as another host of ‘Ca. P. luffae’. This study also showed an example of the same phytoplasma strain ‘Ca. P. luffae’ inducing similar symptoms on different plant hosts.
Identification of phytoplasma can involve the use of other conserved DNA sequence markers for finer differentiation of strains (Duduk and Bertaccini 2011).
These may include the rp gene having more variations than the 16S rRNA, and SecY gene, which may reveal the relationship of phytoplasma strains and geographic distribution (Bertaccini et al. 2014). Application of barcode systems using a fragment of the elongation factor Tu gene has also been used for phytoplasma detection and identification (Makarova et al. 2012). These markers can be used for phytoplasma detection in future studies.
ACKNOWLEDGMENT
The project was supported by AusAID under the project ASEAN-Australia-New Zealand Free Trade Area Economic Cooperation Work Programme (AANZFTA ECWP).
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