Aggressiveness and identification of Pantoea ananatis isolatesABSTRACT
Maize white spot, caused by the bacterium Pantoea ananatis (P. ananatis), occurs in all regions of Brazil, and the use of resistant maize genotypes is recommended for its control. However, to obtain resistant genotypes, the isolates must have their pathogenicity evaluated and the bacterium must be correctly identified. Therefore, the aim of the present study was to assess the aggressiveness of isolates and identify P . ananatis by polymerase chain reaction (PCR) using specific primers. For aggressiveness assessment, nine P. ananatis isolates were inoculated into three different maize genotypes (DKB230, AG9025, and 2B810PW), adopting four replicates. The severity of maize white spot was assessed 2, 4, 6 and 8 days after inoculation, based on a grading scale, and the area under the disease progress curve was calculated. To identify the bacterium, 17 isolates were amplified by PCR using the specific primer pair ANAF/ANAR. Isolates UFU A18 and UFU B13 were the most aggressive to genotypes DKB230 and AG9025. All 17 isolates were identified as P . ananatis. The present study may assist genetic breeding programs in the selection of maize hybrids resistant to this bacterium.
Keywords
disease; white spot; PCR;
Zea mays
RESUMO
A mancha branca do milho, causada pela bactéria Pantoea ananatis ocorre em todas as regiões do Brasil e para o controle da doença recomenda-se a utilização de genótipos resistentes. No entanto, para a obtenção desses deve-se avaliar a patogenicidade no hospedeiro dos isolados a serem utilizados e a correta identificação da bactéria. Portanto, o objetivo deste trabalho foi avaliar a agressividade de isolados e a identificação de P. ananatis, por PCR usando primers específicos. Para a avaliação da virulência foram utilizados nove isolados de P. ananatis inoculados em três genótipos de milho (DKB230, AG9025 e 2B810PW), com quatro repetições. A severidade da mancha branca do milho foi avaliada aos 2, 4, 6 e 8 dias após a inoculação, usando uma escala de notas e calculada a área abaixo da curva de progresso da doença. Para a identificação da bactéria, 17 isolados foram amplificados por PCR usando o par de primers específicos ANAF/ANAR. Os isolados UFU A18 e UFU B13 foram os mais agressivos, para os genótipos DKB230 e AG9025. Os 17 isolados foram identificados como P. ananatis. O presente trabalho poderá auxiliar os programas de melhoramento genético, na seleção de híbridos de milho resistentes à bactéria.
Palavras-chave
Doença; mancha branca; PCR;
Zea mays
Maize white spot caused by the bacterium Pantoea ananatis (10) is the major leaf disease affecting the maize crop (4). Initially, it was described to be caused by the fungus Phaeosphaeria maydis (5); however, Paccola-Meirelles et al. (11) isolated the bacterium from early-stage lesions of leaf spot, identifying it as Pantoea ananatis.
This disease occurs in all regions of Brazil, where yield losses can reach over 60% owing to the use of susceptible genotypes (3, 4). It develops under moderate temperatures, around 14 °C, and high moisture conditions, particularly during frequent rainfall (3). Symptoms are characterized by watery, elliptical-to-circular lesions, similar to anasarca, which initially appear as light-green spots and become straw-colored and necrotic at the crop cycle end. The spot diameter and incidence on the leaves may vary (11). Symptoms begin on the lower leaves of maize plants, rapidly spread to the apex and become more severe after the bolting stage (6).
Fungicides have shown low efficiency in controlling this disease (4); thus, employing resistant varieties is the most effective management strategy due to their feasibility, safety and minimal environmental impact (14). During the development of such varieties in genetic breeding programs, considering the aggressiveness of isolates inoculated into the host and correctly identifying the bacterium is critical. Pantoea ananatis can be identified via biochemical, physiological (12) and molecular tests using the specific primers ANAF/ANAR (1, 11). Therefore, the aims of the current study were to assess the aggressiveness of isolates and identify P. ananatis by PCR.
The isolates of P. ananatis (Table 1) from the working collection of the Laboratory of Plant Bacteriology of Federal University of Uberlândia (UFU), Brazil, were cultivated in the culture medium 523 (9). The bacterial suspension was quantified and calibrated using a spectrophotometer (OD550 = 0, 5, 108 CFU mL-1).
To assess the aggressiveness of P. ananatis isolates on maize genotypes, the experiment was conducted using a randomized block design in a (9 × 3) factorial arrangement, which consisted of nine bacterial isolates (UFU A18, UFU B13, UFU D14, UFU E4, UFU E43, UFU F40, UFU F49, UFU G13, and UFU G77) and three maize genotypes (susceptible DKB230, susceptible AGV9025, and resistant 2B810PW), including six replicates. The seeds were obtained from Dow AgroSciences (Brazil).
Maize seeds were sown in 500g pots containing a mixture of soil, coarse sand and vermiculite, at a 3:1:1 ratio, and two plants per pot. Plants with three–four leaves (approximately 15 days after planting) were sprayed with the bacterial suspension (concentration of 108 CFU mL-1). They were maintained in a moist chamber for 24 h before and after inoculation.
The severity of leaf white spot was assessed at 2, 4, 6 and 8 days after inoculation, using a grading scale from 0 to 4, where 0 = no symptoms; 1 = 1%–25% damage; 2 = 26%–50% damage; 3 = 51%–75% damage; and 4 = over 75% damage (14).
The area under the disease progress curve (AUDPC) was calculated using the formula: AUDPC = ∑ [(Yi + Yi+1)/2] (ti+1 – ti), where ‘Y’ is the disease intensity; ‘t’ is the time (days, interval between evaluations), and ‘i’ is the number of evaluations over time (13). The obtained data underwent analysis of variance, and means were compared according to Tukey’s test, at 0.05 significance level, using SISVAR software (7).
The DNA from 17 P. ananatis isolates was extracted using the Wizard Genomic DNA Purification Kit (Promega, WI, USA) and quantified (ng/μl) with NanoDrop 2000 (Thermo Fisher Scientific, MA, USA). Molecular identification of the isolates was obtained with a primer pair specific for P. ananatis, ANAF (5’-CGT GAA ACT ACC CGT GTC TGT TGC-3’) and ANAR (5’-TGC CAG GGC ATC CAC CGT GTA CGC T-3’), which allowed the amplification of a 380bp fragment (8). Polymerase Chain Reactions (PCR) were performed in a final reaction volume of 12.5 μL, containing 50 ng genomic DNA, 1× PCR buffer with Mg2, 2.0 μM each primer, 0.2 mM dNTP, and 1 U Taq DNA polymerase. The amplification process was carried out in a ThermoHybaid Omn-E thermocycler under the following conditions: one cycle of DNA denaturation at 94ºC for 1 min, followed by 30 cycles of 1 min at 94ºC (extension), 1 min at 68ºC (annealing), 1 min at 72ºC (extension), and a final cycle of 10 min at 72ºC. The amplification products were analyzed using 1.0% agarose gel electrophoresis, stained with SYBR Safe, visualized under ultraviolet light, and photographed.
The AUDPC of maize white spot (Table 2) indicated a significant interaction between P. ananatis isolates and the genotypes. For 2B810PW, which was considered a resistant genotype, no disease occurred. In contrast, the susceptible genotypes DKB230 and AGV9025 demonstrated the presence of white spots. The isolates UFU A18 and UFU B13 were the most aggressive and significantly differed from the other isolates in both susceptible maize genotypes (DKB230 and AGV9025). The bacterial isolates UFU F40 and UFU F49 showed intermediate aggressiveness, whereas UFU D14, UFU E4, UFU E43, UFU G13, and UFU G75 were the least aggressive isolates. No correlation was found between the aggressiveness of isolates and their geographical origin.
Area under the disease progress curve of maize white spot, from different genotypes and different isolates of Pantoea ananatis.
Means followed by different lowercase letters on the row and uppercase letters in the column are significantly different, according to Tukey’s test (P=0.05).
Varied aggressiveness suggest that the pathogen may have distinct races with different levels of virulence each. The evolution of these races may be favored by changes in the pathogen population due to selection pressures, which could be intensified via increased inoculum sources in the field caused by successive planting of the first and second crops, absence of crop rotation, and frequent fungicide application (2). However, P . ananatis races have not been described in maize cultivation in Brazil (14).
In the present study, the severity of white spot was higher in susceptible genotypes, to which this disease has caused significant production losses in maize crops (3). The aggressiveness of isolates is an important parameter to be considered in resistant variety development, since using less aggressive isolates may mask genotype reactions and hinder proper separation of resistant and susceptible isolates. Furthermore, chemical products have proven ineffective in controlling maize white spot, highlighting the need for resistant genotypes (2) to manage the disease.
Seventeen bacterial isolates were amplified using the primer pair ANAF/ANAR, specific to P. ananatis, which resulted in a fragment of approximately 380 bp, confirming the bacterium identity. This bacterium was also identified by PCR in maize, sorghum, sourgrass (Digitaria sp.) (8), and southern sandbur (Cenchrus echinatus) (10). Learning the alternative hosts of P. ananatis allows careful eradication of weeds in the field, preventing them from becoming a source of primary bacterial inoculum to the next crop season, particularly in the second-crop maize cultivation (10).
Adopting resistant varieties is the most effective disease management strategy. The identification of P. ananatis and the use of isolates with different virulence levels may assist genetic breeding programs in selecting resistant bacterial hybrids.
The current study showed that P. ananatis isolates UFU A18 and UFU B13 were more aggressive in susceptible maize genotypes (DKB230 and AGV9025), while the genotype 2B810PW was resistant to the bacterium. The specific primer pair, ANAF/ANAR, allowed the identification of all 17 isolates as P. ananatis.
ACKNOWLEDGMENTS
The first author thanks CAPES for the scholarship, and NDT thanks CNPq for the research productivity scholarship granting.
Declaração de disponibilidade de dados
Os dados de pesquisa estão disponíveis no corpo do artigo.
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Edited by
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EDITOR CIENTÍFICO:
Edson Luiz Furtado https://orcid.org/0000-0002-6924-835X
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EDITOR ASSOCIADO:
Rita de Cassia Panizzi https://orcid.org/0000-0002-0289-6563
