Multifunctional characteristics of Acinetobacter lwoffii Bac109 for growth promotion and colonization in micropropagated sugarcane

Características multifuncionais de Acinetobacter lwoffii Bac109 para promoção de crescimento e colonização em cana-de-açúcar micropropagada

ABSTRACT

Endophytic bacteria with multifunctional characteristics can benefit plants through different mechanisms, as well as promoting growth in an efficient, low-cost and ecofriendly way. This study analyzed the potential of the multifunctional endophytic isolate Acinetobacter lwoffii Bac109 in promoting the early in vitro growth of sugarcane seedlings. The Bac109 strain showed potential to solubilize phosphate in a solid medium (solubilization index: 3.73). In addition, the bacterium was an efficient biocontrol agent against the phytopathogenic fungi Rhizoctonia sp., Fusarium oxysporum, Phoma sp. and Bipolaris papendorfii, showing a performance equal to or better than the commercial antifungal hygromycin B. An in vitro assay confirmed the biofilm production, which increased in the presence of sugarcane root extract. Additionally, A. lwoffii Bac109 showed a strong adhesion to the sugarcane roots. The inoculation of this bacterium in micropropagated sugarcane seedlings increased the shoot length (35 %) and regulated the nonphotochemical energy dissipation after 28 days of cultivation. At the end of the experiment, the bacterium showed a great potential for survival, with 5.72 × 107 CFU mL^-1 recovered from the substrate, what is crucial for plant interaction. The results showed the potential of the biotechnology application for A. lwoffii Bac109 by evaluating multifunctional traits of plant growth promotion and by specific interactions with sugarcane, which may help to improve micropropagation protocols for this crop.

KEYWORDS:
Saccharum sp; biofilm; biocontrol; cross-inoculation; plant growth promoting bacteria

RESUMO

Bactérias endofíticas com características multifuncionais podem beneficiar as plantas por meio de diferentes mecanismos e promover o crescimento de forma eficiente, econômica e ecologicamente correta. Neste estudo, foi investigado o potencial do isolado endofítico multifuncional Acinetobacter lwoffii Bac109, com o objetivo de promover o crescimento de mudas de cana-de-açúcar in vitro. A linhagem Bac109 demonstrou capacidade de solubilização de fosfato em meio sólido (índice de solubilização: 3,73). Além disso, a bactéria foi um eficiente agente de biocontrole contra os fungos fitopatogênicos Rhizoctonia sp., Fusarium oxysporum, Phoma sp. e Bipolaris papendorfii, apresentando desempenho superior ou igual ao antifúngico comercial higromicina B. A produção de biofilme foi confirmada em ensaio in vitro e foi maior na presença de extrato de raiz de cana-de-açúcar. Além disso, A. lwoffii Bac109 apresentou forte adesão às raízes da cana-de-açúcar. A inoculação da bactéria em mudas micropropagadas de cana-de-açúcar aumentou o comprimento da parte aérea (35 %) e a dissipação de energia não fotoquímica regulada após 28 dias de cultivo. Ao término do experimento, a bactéria apresentou grande potencial de sobrevivência, com 5,72 × 10⁷ UFC mL^-1 recuperadas do substrato, o que é crucial para a interação da planta. Os resultados mostraram potencial de aplicação biotecnológica para A. lwoffii Bac109 pela avaliação dos traços multifuncionais para a promoção do crescimento vegetal e por interações específicas com a cana-de-açúcar, as quais podem auxiliar no aprimoramento de protocolos de micropropagação da cultura.

PALAVRAS-CHAVE:
Saccharum sp; biofilme; biocontrole; inoculação cruzada; bactérias promotoras de crescimento de plantas

INTRODUCTION

Brazil is the world’s largest producer and exporter of sugarcane (Saccharum sp.) (Conab 2019). This crop is of great importance to the Brazilian economy, generating millions of dollars annually through the production of sugar, ethanol, bagasse and other derivatives (Barnabas et al. 2015). In this context, researchers have delevoped new sugarcane propagation methods to reduce planting material, improve seedling health and increase yield (Ali et al. 2008).

Micropropagation protocols have been developed for the large scale production of sugarcane, with techniques that allow to obtain uniform clones with high phytosanitary standards, as well as reduced time and space requirements (Kumari et al. 2017). However, their use may affect plant growth promoting microorganisms by reducing the endophytic bacteria population. These bacteria inhabit the interior of plant tissues, and their (re)introduction can benefit plant growth, accelerating its development, protecting against pathogens and reducing production costs (Pereira et al. 2019).

Beneficial plant-microorganism interactions can occur through different mechanisms, such as nutrient solubilization, phytohormone production, nitrogen fixation and production of antimicrobial compounds for pathogen biocontrol (Rohini et al. 2018).

Phytopathogenic fungi like Fusarium sp., Phoma sp., Rhizoctonia sp. and Bipolaris sp. can cause several diseases in sugarcane, generating yield losses (Raza et al. 2019). F. oxysporum causes rot and vascular wilt in several crops, including tomato, onion and cotton (Armitage et al. 2018). Moreover, Rhizoctonia sp. causes damping-off and root rot in a wide range of hosts (Ajayi-Oyetunde & Bradley 2018). In a study in southern China, Bipolaris and Phoma were in the top five common genera of plant pathogenic fungi affecting sugarcane (Raza et al. 2019). Inhibiting the growth of such fungi by bacteria via competition or through the production of bioactive secondary metabolites can benefit plant development by indirectly promoting its growth.

As observed in micropropagated sugarcane (Barreto et al. 2019) and cashew trees (Faria et al. 2021), the introduction of growth-promoting endophytic microorganisms in micropropagated plants can decrease contamination losses and increase seedling survival from their establishment and acclimatization to field planting. This bacterium-plant interaction contributes to sustainable agriculture, since it minimizes the use of inducing compounds and chemical pesticides, also resulting in economic gains for the producer (Silveira et al. 2018).

Phosphorus is an essential macronutrient for plant growth. However, the element is not readily available in the soil for plants (Lobo et al. 2019). Thus, the use of phosphorus-solubilizing microorganisms can be a useful tool for agriculture to reduce the use of mineral fertilizers. Several bacterial genera can solubilize phosphate (Pande et al. 2017), with the genus Acinetobacter standing out for having multiple functional traits for plant growth promotion. These gram-negative bacteria can colonize diverse habitats, including water, soil, plants, animals and humans (Nemec et al. 2019).

In addition to phosphate solubilization, these bacteria act in the production of indole-3-acetic acid (IAA); biological nitrogen fixation (Kuan et al. 2016); production of siderophores and biocontrol against plant pathogens (Zhao et al. 2014); synthesis of the enzyme ACC (1-aminocyclopropane-1-carboxylate) deaminase, thus reducing ethylene levels in plants to mitigate adverse stresses while indirectly promoting plant growth (Ahemad & Kibret 2014); and growth promotion of monocots and dicotyledons (Zhang et al. 2017).

Several studies have shown that bacteria of the genus Acinetobacter have multiple functional traits for plant growth promotion, such as calcium phosphate solubilization, siderophore production, antibiosis and auxin synthesis (Rokhbakhsh-Zamin et al. 2011, Oliveira-Longatti et al. 2014, Kandel et al. 2017). Acinetobacter occurs in association with the rhizosphere and as endophytic bacteria of sugarcane roots, stems and leaves (Velázquez et al. 2008). This indicates the existence of a bacterium-plant interaction that developed throughout evolutionary history (Awais et al. 2017, Santos et al. 2017, Armanhi et al. 2018). However, A. lwoffii Bac109 has never been tested for its ability to promote sugarcane growth.

The Acinetobacter lwoffii strain Bac109 is an endophytic isolate from the roots of Anacardium othonianum Rizzini (Faria et al. 2021). This plant, native to Brazil and popularly known as Cerrado cashew, has a great biotechnological potential for plant growth promotion.

Considering the potential of Acinetobacter in promoting plant growth, the inoculation of this bacterium into sugarcane may represent a viable and fast alternative for the sustainable development of this crop. Thus, the hypothesis of the present study is that the A. lwoffii strain Bac109 has growth-promoting characteristics that can improve the in vitro development of sugarcane. In this context, the study evaluated the functional traits and potential of the strain Bac109 for promoting growth in sugarcane seedlings.

MATERIAL AND METHODS

The A. lwoffii strain Bac109 used in the present study was isolated by Faria et al. (2021) and belongs to the collection of the Instituto Federal Goiano, Rio Verde, Goiás state, Brazil (17º48’15.9”S, 50º54’19.5”W). The sugarcane plants (cultivar CTC 04) used in this study were collected at 14 months of field cultivation at the São Martinho Mill (Quirinópolis, Goiás state, Brazil).

The antagonistic capacity of the bacterial strain Bac109 was tested against the phytopathogenic fungi Rhizoctonia sp., Fusarium oxysporum, Phoma sp. and Bipolaris papendorfii, which belong to the collection of the Instituto Federal Goiano. The bacterial culture was grown in nutrient broth and adjusted to OD₆₀₀ 1.0. Phytopathogenic fungi were inoculated and incubated with the bacteria as proposed by Felestrino et al. (2017).

The Bac109 and sugarcane roots were prepared according to Paungfoo-Lonhienne et al. (2016). The controls consisted of media with PBS or root extract without the bacterium. The plates were incubated for 48 h, at 28 ºC, with shaking at 3 g. Biofilm formation was evaluated by staining with 0.1 % crystal violet. The measurement of the OD₅₉₅ value followed the method by Schembri & Klemm (2001). The assessment of the adhesion of Bac109 to sugarcane roots followed Hozore & Alexander (1991), with 2.48 x 10⁸ CFU mL^-1.

Sugarcane apices were removed and disinfected according to Dutra et al. (2011). After disinfection, the explants were inoculated into test tubes containing 50 % MS medium (Murashige & Skoog 1962) supplemented with 0.22 mg L^-1 of 6-benzylaminopurine (BAP) and 0.11 mg L^-1 of kinetin. The test tubes were kept in the dark for five days, being then kept at 25 ± 2 ºC, with a 16-hour photoperiod and a photon flow density of 27 mmol m^-2 s^-1. Subcultures were performed every 30 days, for 11 months.

The inoculation experiment lasted from May 01 to May 29, 2017. In the experiment, sugarcane seedlings were kept under micropropagation conditions with the bacterial isolate Bac109 in autoclaved sand. The seedlings were cut such that each had shoots of 4 cm and roots of 0.5 cm. At 10 days after subculture, the seedlings were placed in magenta vessels containing sterile sand. The in vitro plant growth bioassay had a completely randomized design and consisted of two treatments (with Bac109 inoculation - “Bac109”; and without bacterial inoculation - “control”), with four replicates each, with a plot consisting of two seedlings.

The nutrient solution (Hoagland & Arnon 1939) was used without phosphate salts and with the addition of inorganic calcium phosphate for plant nutrition. Moreover, the Bac109 treatments received 500 µL of saline solution containing 1.0 x 10⁸ CFU mL^-1 of the bacterial strain per sugarcane seedling, by drenching. When the field capacity of the substrate reached 80 %, the seedlings were irrigated with sterile distilled water for the substrate to return to 100 % of field capacity. To this end, the vessels were weighed every two days and maintained in plant growth chambers (Fitotron, SGC 120) at 26 ºC, with 16-h photoperiod, 80 % humidity and light intensity of 50 µmol m^-2 s^-1.

Biometric evaluations were performed 28 days after the inoculation and consisted of measurements of the number of leaves, shoot length, leaf area and root length. The leaf area was measured from images that were processed using the ImageJ software (Schneider et al. 2012). Subsequently, the seedlings were dried in a forced-air oven at 65 ºC to a constant weight, to determine the shoot and root dry weight.

For the determination of inoculum survival, the total bacteria of the substrate were reisolated. Samples (100 mg) of sand near the roots were collected from each treatment 24 h after planting. Serial dilutions of the soil samples were plated on nutrient agar and incubated at 28 ºC, to enumerate the CFUs. A new reisolation was performed at the end of the experiment (28 days).

To confirm the ability of the reisolated bacteria to solubilize calcium phosphate, 10 µL of the 10^-2 dilution were inoculated in triplicate onto GL medium plates (10 g L^-1 of glucose, 0.05 g L^-1 of yeast extract and 15 g L^-1 of agar), supplemented with CaHPO₄. The latter was generated by the addition of 50 mL of a 10 % K₂HPO₄ solution and 100 mL of a 10 % CaCl₂ solution. The total phosphorus added to the culture media corresponded to 1.85 g L^-1, in the form of insoluble phosphates. The solubilization capacity was determined after three days of incubation at 28 ºC by the formation of a clear halo around the colony (Katznelson & Bose 1959). The experiment was conducted in triplicate and the solubilization index (SI) was calculated according to the formula: SI = halo diameter (cm)/colony diameter (cm).

Chlorophyll a fluorescence was assessed using an Imaging-PAM fluorometer and the Imaging Win software (Heinz Walz, Effeltrich, Germany). For this assay, the plants were dark-adapted for at least 30 min; then, 5 cm of the central region of the apical sugarcane leaves were used for analysis. Fluorescence signals at all points of the analyzed leaf area were captured using a charge-coupled device camera. Fluorescence parameters of chlorophyll a were determined according to Oxborough & Baker (1997). The calculation of the regulated nonphotochemical energy dissipation [Y (NPQ) = (F/F_m’) - (F/F_m)] followed Genty et al. (1989). Five replicates were used for this evaluation.

Antagonism, motility and biofilm production tests were performed in triplicate and statistically analyzed using Anova (GraphPad Prism v. 7.03). Biometric data were subjected to the Shapiro-Wilk normality test, using the Sisvar 5.6 software (Ferreira 2011). All analyses were followed by the Tukey test, with p < 0.05 considered significant.

RESULTS AND DISCUSSION

A. lwoffii Bac109 showed a biocontrol potential against all the phytopathogens under study (Figure 1). The bacterium inhibited the growth of the fungus Rhizoctonia sp. by 27.59 %, in relation to the control (Figures 1A and 1B), whereas hygromycin B inhibited 10.02 % of the fungal growth. Thus, A. lwoffii Bac109 significantly inhibited this fungus (more than two-fold), in relation to the commercial antifungal (p < 0.01). Moreover, Bac109 and hygromycin B inhibited the F. oxysporum growth by 32.28 and 18.74 %, respectively (p < 0.001) (Figures 1A and 1C). The bacterium also inhibited the growth of the fungus Phoma sp. by 22.12 %, which was statistically equal to the fungal inhibition caused by the antibiotic (19.25 %) (Figures 1A and 1D). For the phytopathogen B. papendorfii, the growth inhibition by A. lwoffii (34.69 %) was almost four times greater than that by hygromycin B (8.85 %) (p < 0.0001) (Figures 1A and 1E).

The coculture assay of A. lwoffii Bac109 with the phytopathogenic fungi Rhizoctonia sp., F. oxysporum, Phoma sp. and B. papendorfii showed inhibition of fungal growth in relation to the control without bacteria, which was superior or similar to the inhibition caused by the commercial antifungal hygromycin B. Rokhbakhsh-Zamin et al. (2011) had previously shown the biocontrol potential of Acinetobacter sp. In their study, 27 bacterial strains of this genus, isolated from the rhizosphere of Pennisetum glaucum, inhibited the growth of F. oxysporum by 17-86 %. This in vitro result for fungal control by Bac109 is an indicative of a plant inoculant.

Biofilm production is a mechanism that bacteria use to colonize plant tissues. Biofilms consist of bacterial communities embedded in a matrix composed of exopolysaccharides, DNA and proteins (Meneses et al. 2011, Paungfoo-Lonhienne et al. 2016). Bacterial biofilms on plant surfaces may promote plant growth, protection or pathogenesis (Bogino et al. 2013). The ability of A. lwoffii Bac109 to form biofilms was tested in the absence and presence of sugarcane root extract (Figure 2).

Bac109 produced biofilm in LB. However, the treatment showed a significantly denser crystal violet staining when the bacterium grew in a medium supplemented with sugarcane extract than in the absence of the extract (p < 0.01). This result indicates that A. lwoffii interacts with sugarcane and that the plant extract induces the formation of even more biofilm by the bacterium. This demonstrates the colonization ability of A. lwoffii amongst microorganism-plant interactions. Syed-Ab-Rahman et al. (2018) have also reported the biofilm capacity of Acinetobacter sp.

The results of the assays evaluating the adherence of Acinetobacter to sugarcane roots demonstrated that an average of 3.54 × 10⁹ and 1.99 × 10⁵ CFU mL^-1 of the isolate weakly and strongly adhered to the roots, respectively, confirming the interaction between A. lwoffii and sugarcane. Researches in the field have aimed to find the efficiency of endophytic bacteria inoculants for application in sugarcane (Naveed et al. 2014, Bach et al. 2016). Among the factors considered in these studies, bacterial adherence to roots is an important characteristic for predicting the ability of a microorganism to colonize roots. A. lwoffii Bac109 bacterial cells strongly adhered to sugarcane roots, demonstrating their colonization ability, as well as microorganism-plant interactions. These results agree with those of the biofilm production assay, which revealed an increased A. lwoffii Bac109 biofilm formation in the presence of sugarcane root extract.

After 28 days of cultivation, the sugarcane seedlings inoculated with the Bac109 strain increased their shoot length by 35 %, in relation to the control treatment (Figures 3A and 3B), what may be a differential feature for better development in an acclimatization phase. This result may be due to the auxin biosynthesis, since A. lwoffii Bac109 produces IAA, responsible for promoting cell elongation and increasing the length and number of root hairs (Faria et al. 2021). However, neither the shoot dry weight and shoot/root ratio nor other biometric parameters differed between the treatments. According to the literature, Acinetobacter sp. RSC9 also improves sugarcane growth parameters such as plant height, number of leaves, and shoot and root fresh and dry weight (Patel et al. 2021), demonstrating the ability of the bacteria from this genus to promote plant growth.

Figure 1
Phytopathogen inhibition analysis. A) percentage of phytopathogenic fungi inhibition in the presence of the bacterium Acinetobacter lwoffii strain Bac109 and hygromycin B, relatively to the fungi alone in the control plates. Error bars represent the standard deviation. Asterisks above the lines indicate a significant difference between the bacterial and antibiotic treatments (** p < 0.001; **** p < 0.00001). Tests without hygromycin B: B) Rhizoctonia sp. control plate and Rhizoctonia versus Bac109; C) Fusarium oxysporum control plate and Fusarium versus Bac109; D) Phoma sp. control plate and Phoma versus Bac109; E) Bipolaris papendorfii control plate and Bipolaris versus Bac109.

For the physiological parameters, the nonphotochemical quenching [Y (NPQ)] was higher in inoculated seedlings than in control treatments (Figures 3C and 3D). Chlorophyll a fluorescence, minimum (F₀ )and maximum (F_m )fluorescence and maximum quantum efficiency of photosystem II (Fv/Fm), as well other analyzed parameters, did not differ between the treatments.

Figure 2
Biofilm formation assay for Acinetobacter lwoffii Bac109 cultured in LB broth in the absence or presence of sugarcane root extract. Bars represent the standard deviation. * Significant difference at p < 0.01.

Figure 3
Micropropagated sugarcane seedlings after 28 days of cultivation in chambers inoculated with Acinetobacter lwoffii Bac109 (+Bac) or without bacterium (-Bac/Control). A) Seedlings in growth chambers (bar scale: 3.55 cm); B) biometric data: shoot and root length; C) chlorophyll a fluorescence [F₀: minimal fluorescence; Fv/Fm: maximum quantum efficiency; Y (NPQ): regulated nonphotochemical energy dissipation]. The color code, from 0 (black) to 1 (pink), is shown at the bottom of the image; D) F₀, Fv/Fm and Y (NPQ) data. Means followed by the same letter do not differ by the Tukey test at 0.05 of probability.

The physiological performance evaluation of micropropagated sugarcane seedlings showed that the plants did not have a functional photosynthetic apparatus in all treatments. This characteristic is common for micropropagated plants because the plants are in an environment that favors their growth, limiting the development of the photosynthetic apparatus (Matysiak & Gabryszewska 2016). This characteristic is a major constraint for the adaptation of plants to greenhouse and field conditions. For this reason, the inoculation of beneficial microorganisms before acclimatization could aid in adapting plants to the conditions of the in vivo environment.

Despite the evaluations being conducted under in vitro conditions, the seedlings showed higher rates of nonphotochemical quenching when inoculated with Bac109. This result could suggest an increase in energy dissipation that improves plant development in a new condition, such as a substrate. The photosystem II efficiency, including NPQ, increased in pepper plants inoculated with Bacillus spp., suggesting a better way of disposing of excess light energy, in relation to uninoculated plants (Samaniego-Gámez et al. 2016). Furthermore, the interaction between endophytic bacteria and sugar beets (Beta vulgaris L.) improves the photosynthetic capacity of the crop, increasing the carbohydrate synthesis (Shi et al. 2010). In the present study, the sugarcane seedling with higher nonphotochemical quenching also showed an increase in shoot length that may be a consequence of a higher photosynthesis rate.

In addition, the A. lwoffii survival was tested at 24 hours after the inoculation and at the end of the experiment by reisolation of the bacteria from the substrate. Counts were 1.53 × 10⁸ and 5.72 × 10⁷ CFU mL^-1 of A. lwoffii Bac109, respectively, confirming the ability of the isolate to survive throughout the experiment. Besides presenting characteristics of plant growth-promoting bacteria, the survival, abundance and persistence of the microorganism in the substrate or plant are fundamental characteristics for the development and establishment of an inoculant for crops (Ambrosini et al. 2016).

Reisolated colonies were semiquantitatively tested for the ability to solubilize phosphate. The results showed that the strain maintained this functional trait, with a mean solubilization halo, colony diameter and SI of 3.11 cm, 0.883 mm and 3.73, respectively.

Phosphate solubilization by microorganisms is a mechanism that directly promotes plant growth. This is because the microorganisms that have this ability can make phosphorus available to the plants from insoluble phosphate, contributing to the plant development (Zeng et al. 2017). A report in the literature shows that Acinetobacter sp. UFLA 03-09 solubilizes approximately 20 mg mL^-1 of CaHPO₄ in a liquid medium via acidification of the medium, with a solubilization index similar to that of the Bac109 (3.73) (Marra et al. 2012).

The amount of phosphate solubilized by members of the genus Acinetobacter may vary among strains. For example, Acinetobacter calcoaceticus D10 solubilized 10.07 mg mL^-1 of CaHPO₄ (Zhao et al. 2014), while Acinetobacter sp. ST02 solubilized a maximum of 329.9 μg mL^-1 of phosphate (Ogut et al. 2010). According to these data, A. lwoffii Bac109 stands out within the genus as being highly efficient in phosphate solubilization, improving fertilization by making this nutrient available to plants.

CONCLUSIONS

1. Acinetobacter lwoffii Bac109 presents multiple functional traits to promote in vitro plant growth. The strain solubilizes inorganic phosphate, presenting an efficient biocontrol against phytopathogenic fungi and increasing biofilm production and the initial sugarcane growth;

2. The isolate adheres to sugarcane roots, allowing the population to survive under micropropagation conditions;

3. The combination of the beneficial traits of the Bac109 strain and its ability to efficiently colonize the plant shows a potential for formulating an inoculant that contributes to sustainable agriculture from the early stages of in vitro multiplication.

ACKNOWLEDGMENTS

To the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG) and Instituto Federal Goiano, for their financial support.

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Publication Dates

• Publication in this collection
  01 Dec 2021
• Date of issue
  2021

History

• Received
  21 June 2021
• Accepted
  31 Aug 2021
• Accepted
  14 Oct 2021