Diverse phytoplasmas associated with maize bushy stunt disease in Peru

Corn plants showing symptoms of midribs chlorosis, and leaf reddening, short internodes, ear proliferation, and plant growth reduction were collected in Peru from fields in nine localities in the provinces of Huancayo, Chupaca, and Jauja in the Junín region, and tested to verify phytoplasma presence and identity. Primers amplifying the phytoplasma ribosomal 16S and ribosomal protein genes were used. The phytoplasma presence was detected in symptomatic samples from all the surveyed areas. The sequencing of the obtained amplicons indicated the presence of ‘Candidatus Phytoplasma asteris’ and ‘Ca. P. pruni’-related strains. A BLASTn search of sequenced genes showed that the two ‘Candidatus Phytoplasma’ strains identified in corn shares 100% and 99.82% identity with the ‘Ca. P. asteris’ strains from maize and 99.92% and 99.55% with ‘Ca. P. pruni’-related strains, respectively. The RFLP analyses allowed to enclose these phytoplasma strains in the 16SrI-B and 16SrIII-J subgroups; however, the two phytoplasmas were, in some cases, present in mixed infection. The 16SrIII-J phytoplasma is for the first time reported associated with the maize bushy stunt disease and this represent a relevant information for the disease epidemiology towards its appropriate management in the affected area.


Introduction
Corn is one of the main species cultivated worldwide (FAO, 2020). In Peru, both hard yellow and starchy corns are grown for human consumption as dry or wet grain (Huamanchumo de la Cuba, 2013). According to the Peruvian Ministry of Agrarian Development and Irrigation, the national production of starchy corn for 2019 was 305,198 tons, (MIDAGRI, 2021). Maize bushy stunt (MBS) is the most important disease that decreases maize production especially in Latin American cultivations, where it can decrease the production and yield of the crop up to 100% (Jones & Medina, 2020). The disease has been reported in the United States, Costa Rica (Harrison et al., 1996), Mexico (Pérez-López et al., 2016), Nicaragua (Hruska et al., 1996), Belize (Henríquez et al., 1999), Colombia (Duduk et al., 2008), and Brazil (Bedendo et al., 1997). In Peru, MBS has been reported in the departments of Ancash and Ayacucho (Nault et al., 1979(Nault et al., , 1981 and in the Junin region (Hodgetts et al., 2009;Nipah et al., 2007). The MBS infected corn plants shows symptoms of midribs chlorosis, and leaf reddening, short internodes, ear proliferation, and plant growth reduction (Bedendo et al., 1997). This disease was reported associated with the presence of 'Candidatus Phytoplasma asteris' (16SrI-B subgroup) (Lee et al., 2004) and known to be transmitted by Dalbulus maidis (Hemiptera: Cicadellidae) (Nault & Delong, 1980). The complete genome of a Brazilian strain of this phytoplasma from corn was recently sequenced (Orlovskis et al., 2017). Phytoplasmas are pleomorphic bacteria with size ranging from 200 to 800 nm. They multiply and survive in isotonic environments including plant phloem and hemolymph of insect vectors  and are classified using molecular tools such as nested polymerase chain reaction (PCR) on the 16S ribosomal gene followed by sequencing or restriction fragment length polymorphism (RFLP) analyses. The two methods are defining 'Candidatus Phytoplasma' species and 16S ribosomal groups, respectively (Bertaccini & Lee, 2018;IRPCM, 2004).
Symptoms associated with MBS were observed more than ten years ago in fields located in the Mantaro Valley (Junín region) (Hodgetts et al., 2009). In the last production campaigns these symptoms were again observed in several fields located in the same area. Considering the increasing yield losses, a survey was carried out to verify the symptom incidence and the identity of the associated phytoplasmas to devise the most appropriate management measures to be adopted to reduce the MBS impact in these corn cultivations.

Materials and methods
S a m p l e c o l l e c t i o n a n d d i s e a s e i n c i d e n c e estimation Whole plants with symptoms of chlorotic streaking, shoot proliferation, anthocyanescence, dwarfism, generalized chlorosis, bud atrophy, and internode shortening (Fig. 1) were collected in the department of Junin-Peru from production fields in the localities of Sicaya, San Agustin de Cajas, Hualhuas, and San Jeronimo located in Huancayo province, Tres de Diciembre, San Juan de Iscos, Ahuac, Chupaca in Chupaca province and El Mantaro in Jauja province (Table 1; Fig. 2), between November 2018 and April 2019. A total of 114 samples from nine different areas were sampled and, after washing under tape water, 2.5 g of leaf vein tissues were stored at −20°C for nucleic acid extraction. Disease incidence estimation was calculated in each area as the proportion of fields with the number of plants showing MBS symptoms to the total number of fields assessed multiplied by 100.
Nucleic acid extraction The DNA extraction from the plant samples was done using a CTAB method (Doyle & Doyle, 1990). For negative control, the DNAs of two symptomless plants collected in asymptomatic fields were extracted. The quality and purity of the extracted DNA were verified in a 1% agarose gel in 1X TAE buffer (40 mM Tris-Acetate, and 1 mM EDTA at pH 8.3), stained with ethidium bromide (10 ng/μl) and visualized with a Gel Doc XR+ Gel Documentation System (Bio-Rad Laboratories, Inc.). Quantification was obtained by NanoDrop One (Thermo Scientific™).
All PCR reactions were carried out in a 25 μL mix consisting of 2 μL of each primer (10 μM), 1 μL dNTP mix (100 μM), 2.5 μL buffer (10x), 0.2 μL Taq DNA polymerase (5 U/μL) (Invitrogen, USA) and 2 μL of diluted DNA (approx. 50 ng) with the cycling reported in the corresponding literatures. For the nested PCR amplification, the amplicons were diluted 1: 30 with sterile distilled water and 1 μL was used as a template. The presence of amplified products was verified in 1% agarose gel as described above.
Identification of disease-associated pathogens The 3fwd/3rev, R16F2n/R2, and fU5/rU3 amplicons were subjected to RFLP analyses using Tru1I and/orHhaI (Fermentas, Vilnius, Lithuania) restriction enzymes under the conditions reported by the manufacturer. The obtained restriction profiles were compared with those of 19 phytoplasma strains ( Table 2). The reactions were visualized in 6.7% polyacrylamide gels in 1X TAE buffer. Virtual restriction profiles were produced for 16S rRNA gene sequences of 1290 and 1248 nucleotides in agarose 4% gels using pDRAW32 program and in the iPhyClassifier (Zhao et al., 2009) respectively, in both cases the most informative restriction enzymes (Lee et al., 1998) were used for digestion.
Fragments of the phytoplasma 16S ribosomal and rp genes amplified by MBSF1/MBSR1, fU5/rU3, P1A/ 16SrSR, 3fwd/3rev, rpL2F3/rp(I)R1A, rp(III)F1/ rp(III)R1 and CSSF1/R1 primers were purified from agarose gel using the Wizard® SV Gel and PCR Cleaning System Kit (Promega, USA) following the manufacturer's recommendations. The purified fragments from the positive samples for the 16S ribosomal gene and rp gene were sequenced in both directions with the same primers used for PCR. The sequences obtained were edited using BioEdit (Hall, 1999), assembled and the sequences analyzed using the Basic Local Alignment Search Tool (BLASTn) (Altschul et al., 1990) by comparing them with those deposited in the National Centre of Biotechnology Information (NCBI) GenBank database (https://www.ncbi.nlm.nih.gov/; Benson et al., 2013). The phytoplasma sequences obtained were deposited at the NCBI GenBank database under the accession numbers MW559787, MW578284, M W 5 7 8 2 8 7 , M W 5 7 8 2 8 8 , M W 5 7 8 3 6 3 , a n d MW578290.
Multiple alignments of the consensus sequences were performed using the Cluster W program (Thompson et al., 1994). Phylogenetic analyses were carried out with MEGA v6.0 (Tamura et al., 2013) by the Maximum Likelihood method based on the Kimura 2parameter model (Kimura, 1980) on the 16S rRNA and rp gene sequences. Acholeplasma laidlawii and a 'Ca. P. trifolii' strain were used as outgroups for the two genes, respectively.      Table 1) and phytoplasma controls (as listed in Table 2).  Fig. 3). The use of CSSF1/R1 primers produced in some of the samples amplicons of approximately 700 bp, however their sequences resulted aspecific and did not allow to verify the presence of S. kunkelii. Among the 114 samples 15, from different locations, were sent for sequencing using the P1A/ 16SrSR, R16F2n/R2 or 3fwd/3rev primers used for their amplification and subjected to RFLP analyses. The RFLP and virtual RFLP analyses of these samples with appropriate restriction enzymes (MseI = Tru1I, and HhaI) on the amplicons and the sequences obtained with the phytoplasma primer pairs R16F2n/R2 and 3fwr/3rev, respectively, showed the presence of two profiles and allowed enclosing the detected phytoplasmas in subgroups 16SrI-B and 16SrIII-J (Figs. 4 and 5). A total of five fragments of approximately 1450 bp, amplified by nested PCR with primers P1A/16Sr-SR from samples collected in San Agustin de Cajas (MCl1), San Jerónimo (MSJIV12) and Chupaca (MCHI4, MCCIV2, MCCIII7.1) resulted positive to the presence of 'Ca. P. asteris'. Further amplicon sequencing using the same primers of the sample MCCIII7.1 resulted positive to the presence of a 'Ca. P. pruni'-related strain (Fig. 6). Further amplification and sequencing of the 15 selected samples with ribosomal protein group-specific primers confirmed the presence of the two 'Ca. Phytoplasma' species in mixed infection in two surveyed localities (Chupaca and Ahuac Chupaca, Junin). The BLASTn analysis showed that these sequences, except the one submitted to GenBank under the accession number MW578363, had a 100% identity to those of MBS phytoplasma strains from Colombia, Brazil, and Mexico (GenBank accession numbers HQ530152, CP015149, and AY265208 respectively) and to several other 'Ca. P. asteris'-related strains. The sequence MW578363 showed 99.85% identity to Solanum lycopersicum phytoplasma clone TomRed (GenBank accession number KC412031) from Argentina and to other phytoplasma strains from Colombia, Bolivia, and Brazil resulting 'Ca. P. pruni'-related and 99.92% identity to a Delphinium phytoplasma strain (GenBank accession number EF514210).
The BLASTn analysis of ribosomal gene sequences showed 99.82% and 99.55% identity to the rp gene from 'Ca. P. asteris' strains from maize from Brazil and oil palm from Colombia (GenBank accession numbers CP015149 and KF434318) and to 'Ca. P. pruni'-related strains from Cucurbita maxima from Argentina (GenBank accession number KC412017), respectively. The dendrogram confirmed that these two 'Candidatus Phytoplasma' strains cluster with phytoplasmas mainly identified in South American areas and belonging to the two identified 'Ca. Phytoplasma' species (Fig. 7).

Discussion
The molecular testing performed allowed the detection of phytoplasmas in the majority of the symptomatic corn samples collected in the surveyed growing areas of Central Peru (Table 1). The use of U5/U3 primers in nested PCR assays allowed verifying a widespread presence of phytoplasmas in almost all the fields indicating a very close correspondence between symptoms and phytoplasma presence. The two phytoplasmas identified were not differentiated by corn phytoplasma specific primers MBSF1/R1 (Harrison et al., 1996), however Fig. 6 Molecular analysis by maximum likelihood method of the 16S rRNA gene sequences obtained from the phytoplasmas detected in corn samples from Peru (in bold red). 'Candidatus Phytoplasma' species officially described are used for comparison. Acholeplasma laidlawii is used as an outgroup. The tree with the highest log-likelihood is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA6 Fig. 7 Molecular phylogenetic analysis by Maximum Likelihood of the rp gene sequences obtained from phytoplasmas detected in corn from Peru (in bold blue). The strain from corn was compared with the rp gene of strains of 'Ca. P. asteris' in a) and of 'Ca. P. pruni' in b). 'Ca. P. trifolii' (16SrVI-A), vinca virescence strain VR, is used as an outgroup. Analyses were conducted in MEGA6 RFLP and sequencing of the amplicons obtained with 16S rRNA gene and group specific ribosomal protein primers allowed to confirm the presence of 16SrI-B and 16SrIII-J subgroups, in some cases in mixed infection.
The presence of S. kunkelii was not detected in spite of the use of primers reported to be specific (Barros et al., 2001). This pathogen was identified in screening carried out in Mexico and Brazil (Davis et al., 1972;Chen & Liao, 1975;Gordon et al., 1985;Bedendo et al., 1997) in corn plants showing symptoms very similar to those found in this survey. However, these symptoms are, according to the literature, also associated with the presence of viruses and therefore are not specific and the presence of viruses was not screened since the main focus was the identification of the phytoplasmas that were already reported associated to the disease in this area (Hodgetts et al., 2009).
While the 'Ca. P. asteris' is widely reported to infect corn on the American continent (Lee et al., 2004), this is the first detection of 16SrIII-J phytoplasmas in corn. The 16SrIII-J phytoplasma strain is widespread in other countries of South America such as Brazil (Montano et al., 2000), Argentina (Galdeano et al., 2013) and Chile (Quiroga et al., 2020) where it was detected in several herbaceous and woody horticultural species.
Recently it was also identified in faba bean in Peru (Torres-Suarez et al., 2021) and insect vectors of this phytoplasma strain were identified as Paratanus exitiosus (Beamer) and Bergallia valdiviana Berg 1881 in Chile (Quiroga et al., 2019).
Both phytoplasma strains detected appear to be very closely related to strains detected mainly in Latin America, suggesting a wide distribution of similar strains in different countries and diverse crops. Considering that more than one corn cultivation cycle is performed each year in these areas, it is important to verify insect vectors presence for both phytoplasma strains. Crop rotation and a change in vector feeding preference could also play a relevant role in the infection of the 16SrIII-J phytoplasmas strain in corn. The presence of infected erratic seeds, spouting near the corn fields could maintain and increase the disease dissemination to new crops by insect vectors making very relevant the need of verification of phytoplasma seed transmission reported for corn in other areas of the world (Calari et al., 2011;Satta et al., 2019). The further verification of these epidemiologic aspects will make it possible to develop and apply well focused and environmentally friendly management tools to reduce the epidemic spread of phytoplasmas in corn also in other world cultivations recently reported also in Europe (Çağlar et al., 2019Duduk & Bertaccini, 2006).
Informed consent All the authors declare that the principles of ethical and professional conduct were duly followed during the execution of this research. The research was funded by Vice-Rector's Office for Research (CANON Projects, resolution N°2 771-R-2019) of the Universidad Nacional del Centro del Peru.
Conflict of interest All authors affirm that 1) there exist no actual or potential conflict of interests to disclose, 2) the manuscript is original and has not been published previously (partly or in full), and is not under review for publication elsewhere, 3) all the necessary local, national and international standards, regulations and conventions, including normal scientific ethical practices, have been duly followed and respected. Additionally, all authors have endorsed the final version of the manuscript before submission.
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