Edel Pérez-López

Maize bushy stunt phytoplasma affects native corn at high elevations in Southeast Mexico

In the 2013–2014 growing season, field surveys were conducted in native corn fields located in high altitude agricultural communities in the ‘Sierra Norte de Puebla’ in Mexico. Symptoms typical of maize bushy stunt (MBS) disease were observed and DNA extracted from symptomatic native corn plants was used as template to confirm the presence of phytoplasmas. Amplification and sequencing of 16S rRNA-encoding sequences and chaperonin 60 universal target (cpn60 UT) sequences followed by in vitro restriction fragment length polymorphism and phylogenetic analyses revealed that the phytoplasma detected belongs to the subgroup 16SrI-B, ‘Candidatus Phytoplasma asteris’. Based on 16S rRNA-encoding gene sequence analysis and on a single nucleotide polymorphism within the cpn60 UT sequence, two MBS strains, MBS-Puebla and MBS-Veracruz, were identified. This is the first detection of MBS phytoplasma (MBSP) affecting native corn and the first molecular survey made in corn fields in Mexico to detect and characterize MBSP. We discuss these results in light of the potential evolutionary relationship between corn and MBSP.

Phytoplasma classification and phylogeny based on in silico and in vitro RFLP analysis of cpn60 universal target sequences.

Phytoplasmas are unculturable, phytopathogenic bacteria that cause economic losses worldwide. As unculturable micro-organisms, phytoplasma taxonomy has been based on the use of the 16S rRNA-encoding gene to establish 16Sr groups and subgroups based on the restriction fragment length polymorphism (RFLP) pattern resulting from the digestion of amplicon (in vitro) or sequence (in silico) with seventeen restriction enzymes. Problems such as heterogeneity of the ribosomal operon and the inability to differentiate closely related phytoplasma strains has motivated the search for additional markers capable of providing finer differentiation of phytoplasma strains. In this study we developed and validated a scheme to classify phytoplasmas based on the use of cpn60 universal target (cpn60 UT) sequences. Ninety-six cpn60 UT sequences from strains belonging to 19 16Sr subgroups were subjected to in silico RFLP using pDRAW32 software, resulting in 25 distinctive RFLP profiles. Based on these results we delineated cpn60 UT groups and subgroups, and established a threshold similarity coefficient for groups and subgroups classifying all the strains analysed in this study. The nucleotide identity among the reference strains, the correspondence between in vitro and in silico RFLP, and the phylogenetic relationships of phytoplasma strains based on cpn60 UT sequences are also discussed.

Molecular diagnostic assays based on cpn60 UT sequences reveal the geographic distribution of subgroup 16SrXIII-(A/I)I phytoplasma in Mexico

Geographically diverse samples from strawberry exhibiting symptoms of Strawberry Green Petal (SbGP), periwinkle plants with virescence, and blackberry, blueberry, and raspberry plants displaying yellowing and inedible fruits, were assayed for the presence of phytoplasma DNA. PCR targeting the 16S rRNA-encoding gene and chaperonin-60 (cpn60) showed that the plants were infected with phytoplasma subgroup16SrXIII-(A/I)I (SbGP/MPV). To examine the geographic distribution of this pathogen in Mexico, we designed an array of cpn60-targeted molecular diagnostic assays for SbGP/MPV phytoplasma. A fluorescent microsphere hybridization assay was designed that was capable of detecting SbGP/MPV phytoplasma in infected plant tissues, successfully differentiating it from other known phytoplasma cpn60 UT sequences, while identifying a double infection with SbGP/MPV and aster yellows (16SrI) phytoplasma. Two quantitative assays, quantitative real-time PCR (qRT-PCR) and droplet digital PCR (ddPCR), gave similar results in infected samples. Finally, a loop-mediated isothermal amplification (LAMP) assay provided rapid detection of SbGP/MPV phytoplasma DNA. Application of these assays revealed that SbGP/MPV phytoplasma is widely distributed in Central Mexico, with positive samples identified from eleven localities within three states separated by hundreds of kilometres. These results also provide tools for determining the presence and geographic distribution of this pathogen in plant and insect samples in other localities.

Novel phytoplasma strains of X-disease group unveil genetic markers that distinguish North American and South American geographic lineages within subgroups 16SrIII-J and 16SrIII-U

Phytoplasmas in the X-disease group (16SrIII) are highly diverse in terms of geographic distributions, vectorship and plant host specificity. Such biological and ecological diversity is often correlated with distinctive genetic markers present in evolutionarily conserved genes. Based on restriction fragment length polymorphism (RFLP) markers in the 16S rRNA gene sequences, 29 subgroups have been delineated, with most of them being found in the Americas. However, it has been unknown whether distinct geographic lineages are present within a given subgroup. Prior to this study, phytoplasmas belonging to subgroups 16SrIII-J and 16SrIII-U were reported only in countries located in South America. In the present study, we identified new phytoplasmas strains closely related to the reference strains of the two subgroups in Mexico, a North American country. These newly identified Mexican strains possess unique RFLP, single nucleotide polymorphism, and fragmental deletion markers in 16S rRNA- and/or ribosomal protein-encoding genes. Since these markers consistently distinguished the Mexican strains from their South American counterparts, they may represent emerging or previously unknown North American geographic lineages of the subgroups 16SrIII-J and 16SrIII-U.

Absence of Corn Stunt Spiroplasma and Maize Bushy Stunt Phytoplasma in Leafhoppers (Hemiptera: Cicadellidae) that Inhabit Edge Grasses Throughout Winter in Jalisco, Mexico

Summary Leafhoppers (Hemiptera: Cicadellidae) that inhabit edge grasses throughout the winter were tested for the presence of corn stunt spiroplasma (CSS) and maize bushy stunt phytoplasma (MBSP). Leafhoppers were sampled using a sweep net at least once a week between Feb and Jun 2014 in Jalisco, Mexico. In total, 2,263 leafhoppers from 27 different taxa were collected. Stirellus bicolor Van Duzee and Graminella sonora (Ball) were the most abundant leafhoppers, with smaller numbers of Dalbulus maidis Delong and D. elimatus Ball. None of the leafhoppers tested positive for CSS or MBSP, suggesting that these 2 bacterial pathogens do not overwinter in leafhoppers that live on edge grasses. This is an important finding, because several studies have found that crop edges are beneficial reservoirs of insect predators and parasitoids, including those that attack insect vectors of plant pathogens.

Periwinkle proliferation disease associated with 16SrI-B phytoplasma in Mexico

Catharantus roseus, known as periwinkle, is highly susceptible to phytoplasma infection. Periwinkle plants showing proliferation symptoms were detected during 2013–2014 in four geographically distant states in Mexico. The presence of phytoplasmas was confirmed through the amplification of 16S F2nR2 and cpn60 UT sequences from symptomatic plants. Sequencing, phylogenetic analysis and in vitro RFLP revealed that the isolates were ‘Candidatus Phytoplasma asteris’-related strains and members of the 16SrI-B subgroup, confirming the association of this phytoplasma group with periwinkle proliferation disease in Mexico. We also demonstrated that the use of the approximately 550 pb cpn60 universal target sequences allow the differentiation of two 16SrI-B strains, designated here as MePP-Centre, and MePP-South.

Identification of grass white leaf disease associated with a ‘ Candidatus Phytoplasma asteris’-related phytoplasma strain (16SrI-B and cpn60 I-IIIB) in Mexico

Samples of a wild grass showing white leaf symptoms were collected in 2015 in Veracruz, Mexico around sugarcane plantations. DNA amplification, sequencing and phylogenetic analysis revealed the presence of a ‘Candidatus Phytoplasma asteris’-related strain in the plants that were positive using 16S-based and cpn60 PCR assays. The strain determined to be associated with the grass white leaf disease was identified as a member of the 16SrI-B subgroup and a member of the cpn60 I-IIIB subgroup through in silico RFLP analysis. This is the first report of phytoplasma-infected grass plants associated with white leaf disease in Mexico. The implications of these findings are vital for the management of other plant hosts of the family Poaceae such as sugarcane and corn, and point to weedy grasses as a potential source of phytoplasma inoculum for nearby crop plants.

Detection of Maize Bushy Stunt Phytoplasma in Leafhoppers Collected in Native Corn Crops Grown at High Elevations in Southeast Mexico

Abstract Phytoplasmas are wall-less bacteria, unculturable in vitro, and transmitted primarily by leafhoppers (Cicadellidae). Maize bushy stunt disease has been linked to phytoplasmas belonging to the16SrI-B subgroup and vectored by leafhoppers in the genus Dalbulus spp. (Hemiptera: Cicadellidae). The recent detection of maize bushy stunt affecting native corn, maize, in the southeast highlands of Mexico motivated the survey to determine which leafhoppers were associated with this crop during the 2013-2014 growing season. We detected 7 leafhopper genera in native corn cultivated 2,400 meters above sea level (masl), with 4 of these genera reported for the first time in corn. Based on external morphology and male genitalia, we identified Idiodonus wickhami (Ball) (Hemiptera: Cicadellidae), Amblysellus grex (Oman) (Hemiptera: Cicadellidae), Empoasca fabae (Harris) (Hemiptera: Cicadellidae), Macrosteles quadrilineatus (Forbes) (Hemiptera: Cicadellidae), and Dalbulus elimatus (Ball) (Hemiptera: Cicadellidae). We were not able to identify the leafhopper genera Graphocephala (Hemiptera: Cicadellidae) and Erythridula (Hemiptera: Cicadellidae) to species because of a lack of male leafhoppers. Nymphal stages of I. wickhami also were identified using taxonomic and molecular tools. The presence of adults and nymphs of I. wickhami in the crop suggest that native corn grown in the southeast highlands of Mexico is a feeding and reproductive host for I. wickhami. Moreover, I. wickhami was found infected with 16SrI-B strain maize bushy stunt-Ver while D. elimatus, a well-known maize bushy stunt phytoplasma vector, was found infected with the 16SrI-B strain maize bushy stunt-Pueb.

Maize bushy stunt in native corn: implications for Mexican “subsistence farmers”

Native corn in Mexico can be affected by maize bushy stunt disease (MBS), which can negatively impact maize-farming operations. Maize bushy stunt symptoms in native corn were linked to the presence of phytoplasmas through molecular analysis of symptomatic native corn plants in Mexico. These findings motivated a socioeconomic characterization and identification of the management strategies developed by the “subsistence farmers” of those communities to answer the questions: What elements of farming affect the presence of MBS disease in “subsistence farmers” fields? What do “subsistence farmers” know about MBS disease and the pathogen associated with MBS disease? How best can MBS disease be managed in small farming communities in Mexico? Through face-to-face interviews with growers and the corresponding analyses of their responses, we identified a widespread lack of knowledge about MBS disease, its causal agent and its vector(s). This study highlighted factors useful to guide future management decisions and education programs, as well as further research to transmit information about MBS disease among “subsistence farmers” in rural communities in Mexico.

16SrII phytoplasma associated with date palm and Mexican fan palm in Saudi Arabia

Date palm and Mexican fan palm trees showing symptoms previously associated with phytoplasmas were observed in the Al-Qassim region, Saudi Arabia in 2017. DNA amplification, sequencing, and phylogenetic analysis revealed the presence of a ‘Candidatus Phytoplasma australasia’- related strain in eighteen of the eighty-three symptomatic plants collected that were positive using 16S-based assays. This study confirmed the presence of phytoplasma affecting date palms in Saudi Arabia and it is the first report of several date palm cultivars associated with ‘Ca. P. australasia’-related strains. This is also the first report worldwide of Mexican fan palm trees affected by 16SrII phytoplasma strains and the first report of this plant host affected by phytoplasmas in Saudi Arabia. The implications of these findings are vital to implement management strategies and avoid economic losses in Saudi Arabia and the Middle East, which are the main producers of dates in the world.