Mauricio Luna-Rodríguez

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.

Vanilla rhizobacteria as antagonists against Fusarium oxysporum f. sp. vanillae.

Pathogens such as Fusarium oxysporum strongly affect the health of various agricultural crops like vanilla. However, despite significant economic losses caused by this pathogen there is no efficient method for its control. Therefore, we propose using rhizobacteria obtained from vanilla roots against F. oxysporum f. sp. vanillae. The results showed that there was no positive correlation between the antagonism expressed under in vitro conditions and those expressed under greenhouse conditions. The 16S rDNA gene analysis indicated that the bacterial genera tested corresponded to Sphingobacterium, Staphylococcus, Serratia, Psychrobacter, Pseudomonas and Stenotrophomonas. The in vitro antifungal activity was evaluated using three culture media (Potato Dextrose Agar, Nutrient Agar and Czapek) using the empty box technique (antagonism). Isolates of Staphylococcus xylosus BAC-JAG15, Serratia sp. BAC-JAG4 and Stenotrophomonas sp. BAC-JAG1 showed 90% in vitro antagonism against F. oxysporum in the three media tested. In the greenhouse evaluation, plants treated with these isolates initially showed symptoms of chlorosis without developing characteristic symptoms of disease produced by F. oxysporum f. sp. vanillae. This demonstrates protection provided by rhizobacteria against infection from F. oxysporum f. sp. vanillae in vanilla plants.

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.

Evaluation of molecular variability in germplasm of vanilla ( Vanilla planifolia G. Jackson in Andrews) in Southeast Mexico: implications for genetic improvement and conservation

Molecular variability of vanilla ( Vanilla planifolia ) and three wild species was assessed to explore the possible sources of variation that can be used for crop improvement. A total of 154 ISSR loci were analysed by the UPGMA, assignment tests of individuals (STRUCTURE) and indices of genetic diversity. The assignment tests were done at two levels: first considering the four species and then only the accessions of V. planifolia . The molecular analysis indicated 99.3% polymorphism among all species and 70.45% within V. planifolia . The UPGMA showed the separation of these four species into three groups and grouped V. planifolia accessions into three subgroups. The more genetically differentiated accessions were of the Rayada morphotype and a wild accession was from Oaxaca, followed by a wild accession from Quintana Roo; all the commercial accessions of V. planifolia (Mansa morphotype) were grouped together. The STRUCTURE analysis differentiated between V. planifolia and the three wild species, and among the accessions of the Mansa and Rayada morphotypes and the wild accessions. The STRUCTURE analysis also indicated the presence of mixed individuals. These results are of great importance since the accessions of V. planifolia that are genetically more differentiated are the most threatened due to the scarcity of these individuals, the destruction of habitat and replacement by the commercial morphotype. These individuals should be salvaged and used to expand the genetic background of vanilla.

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.

Influence of season and organ on antibacterial activity ofMagnolia dealbataZucc. against two phytopathogenic bacteria

As an alternative to reduce the use of agrochemicals, plant extracts can be a better choice in the control of phytopathogens. In this study, we tested the effects of Magnolia dealbata Zucc. crude extracts on the bacterial growth of Pectobacterium carotovorum and Pseudomonas cichorii, both micro-organisms of phytopathogenic relevance. In addition, our other aim was to explain the influence of the seasons and source organs at different maturing stages in the bioactivity of the extracts. Several ethyl acetate and ethanol crude extracts obtained from leaves, bark, flowers, polyfollicles and seeds were tested using the disk-diffusion method. The ethyl acetate extract of M. dealbata polyfollicle collected in summer inhibited the development of P. carotovorum and P. cichorii to the same extent as the tetracycline positive control (Z=1.0690, p=.285 and Z=1.278, p=.201, respectively) while the ethyl acetate seed extract inhibited the growth of P. carotovorum, but did not equal the effect of tetracycline (Z=2.022, p=.043). It can be concluded that the extracts obtained during summer showed the highest inhibition of these bacteria.