Reduction in Microbial Manifestation on In Vitro Culture of Eucalyptus Microcorys

Micropropagation is one of the main applications of tissue culture of plants. It allows rejuvenation and, consequently, improves explant rooting of woody species. However, one of its main problems is the risk of microbial manifestation. Here, we aimed to evaluate the survival and the reduction in microbial manifestations on in vitro cultures of Eucalyptus microcorys with the addition of chemical agents (sodium hypochlorite or streptomycin) to WPM culture medium. We used in vitro cultivated explants from adventitious shoots from two E. microcorys mother plants, over at 44 years-old. We drew from the multiplication phase explants with clumps of 8 to 12 shoots that displayed bacterial manifestation and submitted them to microbe control, adding different chemical agents at different concentrations to the culture medium: sodium hypochlorite (NaClO) and the antibiotic streptomycin. The results indicated that the best concentrations to reduce the manifestation of microorganisms on in vitro cultivation were 100 mg L − 1 of streptomycin and sodium hypochlorite with 0.003% of active chlorine, since these concentrations maintained 100% of the explants alive and reduced microbial manifestation more eciently.


Introduction
Trees from the species-rich genera Eucalyptus and Corymbia are among the most cultivated in the world, due to their adaptation to different climate and soil conditions, fast growth rates and a large variety of products, such as wood, cellulose, fodder, biofuel, essential oils and bioactive compounds [1]. Among the several species within these genera, Eucalyptus microcorys F. Muell has favorable traits for silvicultural applications, including moderate resistance to frost, tolerance to re, good regeneration capacity through resprouting, and resistance to wood rot caused by the fungus Gloeophyllum trabeum [2].
However, despite its potential for wood production, E. microcorys has been less employed in forest composition than other eucalypts species, and few scienti c reports are found on the topic [3]. One of the main barriers for propagation is the low regenerative capacity caused by the ontogenetic aging of tissues [4], which reduces regeneration capacity and hinders adventitious rooting [5].
An alternative to overcome the issues related to woody species propagation is micropropagation, which is one of the main applications of plant tissue culture [6]. In addition to its practicality and greater impact for the mass multiplication of tree species [7], micropropagation is an alternative for tissue rejuvenation and, consequently, for the improvement of adventitious rooting in the clones production of Eucalyptus spp. and Corymbia spp. [8,9].
However, microbial manifestation of the cultures, especially by fungi and bacteria, hamper explant multiplication and pose the main limitation for the micropropagation of woody species [10]. Microbial manifestations may arise from different sources, such as infected plant material (endophytic manifestation), inadequate handling of the explants and/or instruments, and precarious preparation of the culture medium or laboratory conditions [11].
Microbial manifestation is one of the main causes of economic loss in commercial and scienti c laboratories devoted to tissue culture; therefore, controlling is essential for successful in vitro cultivation [12]. Accordingly, the prevention or elimination of microbial manifestation from in vitro cultivations has been the focus of several studies, ranging from the development of asepsis protocols and handling of mother plants, to the use of antimicrobial products in the culture medium [13].
A few disinfesting compounds have been used aiming to reduce or eliminate microorganisms from in vitro cultures, including chlorine-based compounds, such as sodium hypochlorite and calcium hypochlorite [14], and antibiotics [15]. Sodium hypochlorite (NaClO) has been widely used as a chemical sterilizer, both for explant asepsis before inoculation and as a supplement in the culture medium of herbaceous and woody species [16,17]. In addition to its low cost, easy acquisition and e ciency, Like sodium hypochlorite, antibiotics can also be incorporated into culture media or used as a pretreatment before explant inoculation in vitro [29,30]. The antibiotic streptomycin, belonging to the group of aminoglycosides, has bactericidal effect for a broad range of gram-negative and gram-positive bacteria and is able to stop the onset of protein synthesis and/or reduce ongoing syntheses [31].
Thus, given the importance of culture medium asepsis and the losses entailed by microbial manifestation in in vitro cultures, we aimed to evaluate the survival and reduction in microbial manifestation in Eucalyptus microcorys explants cultivated in vitro through the addition of sodium hypochlorite and streptomycin to the culture medium.

Source and origin of explants
For the experiments, we used in vitro cultivated explants from Eucalyptus microcorys adventitious shoots. For the multiplication phase, the explants were kept in woody-plant medium (WPM) [39] supplied with 0.5 mg L -1 of BAP (6-benzylaminopurine) and 0.05 mg L -1 of NAA (α-naphthaleneacetic acid). The explants were cultivated in vitro for 18 months, subcultured in intervals of 25 days and kept in a growth room.
These explants cultivated in vitro originated from epicormic shoots from two E. microcorys mother plants, over at 44 years-old. These trees were sourced from a forest stand comprising different origins of Eucalyptus and Corymbia species, established in 1974 on the campus of the Federal University of Lavras (21°22'75" S, 44°96'98" W), in the municipality of Lavras -MG, Brazil.

Preparation of the culture medium for multiplication and in vitro conditions
We prepared the culture medium with distilled water, 6 g L -1 of agar and 20 g L -1 of sucrose (C 12 H 22 O 11 /342.30 g). Before adding the agar, we adjusted the pH of the culture medium to 5.8 with HCl and/or NaOH. The solution was autoclaved at 121°C (~1.0 kgf cm -2 ) for 20 minutes. We added BAP and NAA to the culture medium before autoclaving. We cultivated the explants in a growth room at 24°C (±1°C), with a 16-hour photoperiod and light intensity of 40 µmol m -2 s -1 .

Reduction in microbial manifestation
We drew from the multiplication phase explants with clumps of 8 to 12 shoots that displayed bacterial manifestation and submitted them to microbe control, adding different chemical agents at different concentrations to the culture medium: sodium hypochlorite (NaClO) and the antibiotic streptomycin (Table 1). Before their addition to the autoclaved culture media, we exposed the streptomycin and sodium hypochlorite to ultraviolet light for 15 minutes in a laminar ow chamber. The streptomycin was added to the culture medium with a syringe coupled to a Millipore® lter of 0.22 µm of pore size. The sodium hypochlorite was added with a pipette. All utensils were previously autoclaved. After adding the chemical agents, we homogenized the culture media and poured them into test tubes where the explants were Each treatment had 20 replications and followed a completely randomised design in a factorial arrangement (2 × 6) evaluating the interaction between two individuals of Eucalyptus microcorys and six concentrations of the chemical agents targeted at reducing the manifestation of microorganisms. The explants were kept in a growth room and subcultured every 25 days. 75 days following each treatment, we evaluated the percentage of explant survival and the percentage of reduction in microbial manifestation in the culture medium. The methodological sequence is described in Figure 1.

Statistical analysis
We submitted the collected data to a Hartley test (p > 0.05) to assess variance homogeneity among treatments and to a Shapiro-Wilk test to (p > 0.05) to assess data normality. When needed, the data were Box-Cox transformed to meet normality assumptions. We then performed an analysis of variance (ANOVA, p < 0.05) and, accordingly to its signi cance, compared the data from the qualitative factors through a Scott-Knott test (p < 0.05). We performed these analyses in the environment R version 3.5.2 using the package ExpDes [40].

Results And Discussion
Survival of explants According to the analysis of variance, there was no signi cant effect between the factor individual and survival percentage. Survival only varied signi cantly with the chemical agent used. Additionally, no interaction between individuals and chemical agents was found.
The highest survival percentages of Eucalyptus microcorys (above 87%) were achieved with the addition of sodium hypochlorite at the three concentrations (0.001%; 0.003% or 0.005% of active chlorine) and with the addition of streptomycin at 100 mg L -1 to the culture media. However, when streptomycin was added at 250 mg L -1 and 500 mg L -1 , the percentage of explant survival decreased (Table 2).
In vitro plant production is successful when contamination-related losses do not exceed 2.0% per subculture during micropropagation. According to this, we found satisfactory results with sodium hypochlorite at 0.001% and 0.003%, and streptomycin at 100 mg L -1 [41]. These treatments resulted in explant survival percentages between 95% and 100% after the third subculture.
The chemical sterilization of in vitro culture media with sodium hypochlorite has been tested for several species. The most commonly used concentration is 0.003% of active chlorine. Higher concentrations may be phytotoxic to the cultures, as shown in in vitro cultures of Eucalyptus benthamii and Prunus mume [42]. In Eucalyptus benthamii, active chlorine concentrations above 0.005% reduced the percentage of viable shoots [16]; and in Prunus mume, the addition of 0.001% of active chlorine reduced explant survival percentage [43]. Our results for Eucalyptus microcorys agree with those reported in the literature: the addition of sodium hypochlorite to the culture medium at 0.001% and 0.003% maintained 100% of explants alive, whereas its addition at 0.005% resulted in an explant survival percentage of 87.5%.
In the literature, evaluating the effect of chemical sterilization on the culture medium of in vitro Cochlospermum regium multiplication, we found the highest percentages of explant survival at 0.001% and 0.005% of active chlorine concentration: 75% and 65% of survival, respectively [25]. In Bambusa vulgaris, the highest survival percentage (68%) was found when sodium hypochlorite was added at 0.004% [26]. In this study, we found similar results to those from the literature, with an explant survival percentage of 87.5% after the addition to the culture medium of sodium hypochlorite at 0.005% of active chlorine. In our study, increasing the concentration of active chlorine above 0.003% on the culture medium increased explant mortality, although not signi cantly (Table 2).
Many authors have reported the use of antibiotics to control and/or eliminate microorganisms from in vitro cultures [13,44,45]. But depending on antibiotic concentration and the tolerance of the focal species, antibiotics may also have phytotoxic effects on the culture and alter in vitro plant growth [46,47].
In our study, the use of streptomycin at 250 and 500 mg L -1 reduced the survival percentage of Eucalyptus microcorys explants.
In the literature, we nd reports of a decrease in Solanum tuberosum explant survival with increasing addition of streptomycin in the nutrient medium [34]. At 64 mg L -1 and 128 mg L -1 , the use of streptomycin was followed by explant survival percentages around 90% and 80%; whereas at 1,024 mg L -1 , survival was around 50%. Our results, of decreasing explant survival with increasing streptomycin concentration, agree with the literature. At 100 mg L -1 , the addition of streptomycin was followed by 95% of explant survival; whereas at 250 mg L -1 and 500 mg L -1 , explant survival was 82.5% and 85%, respectively. On in vitro cultures of Persea americana, the addition of streptomycin at concentrations between 12.5 to 200 mg L -1 was shown to have a toxic effect [33]. Therefore, the use of streptomycin would only be recommended in extreme cases, depending on the susceptibility of the manifesting microorganism and the concentration needed to control it. In the micropropagation of Helianthus tuberosus the use of streptomycin above 50 mg L -1 inhibits shoot emission [32]. Conversely, our results showed a satisfactory response of Eucalyptus microcorys to the addition of streptomycin at 100 mg L -1 , which resulted in an average explant survival of 95%.

Reduction of microbial manifestation
The analysis of variance revealed a signi cant effect between individuals and the reduction in microbial manifestation. The chemical agent used had a signi cant effect on the reduction of microbial manifestation, and a signi cant interaction was found between individual and chemical agent. Individual 1 displayed statistically undistinguishable responses to all chemical agents and concentrations in terms of reduction in microbial manifestation. But individual 2 displayed statistically different responses depending on the chemical agent and its concentration ( Table 3).
The culture medium supplied with streptomycin at 100 mg L -1 displayed the best result: it reduced microbial manifestation in 65% of individual 2 explants and 15% of individual 1 explants. The addition of sodium hypochlorite at 0.003% of active chlorine reduced microbial manifestation in 25% of individual 2 explants and 5% of individual 1 explants. The other treatments did not contribute to reduce microbial manifestation in either individual (Table 3).
In addition to their better responses to the use of chemical agents for reducing microbial manifestation, explants from individual 2 were also more vigorous. In general, the success of in vitro cultures may vary depending on the genetic material, growing conditions, type of explant, asepsis, phytotoxicity and physiological aspects of the mother plant [16]. The latter aspect was likely responsible for the difference in vigor that we found in the explants of the two individuals (or mother plants). The different physiological conditions of the mother plants probably in uenced the physical aspects of the explants and their responses in terms of the reduction in microbial manifestation.
In [38]. In Vitis vinifera, the addition of streptomycin at 100 mg L -1 did not control the manifestation of bacterial canker, and after 40 days of in vitro cultivation, 100% of the explants remained with the bacterium [37]. Unlike these reports, our results for Eucalyptus microcorys showed that the addition of streptomycin at 250 mg L -1 did not reduce microbial manifestation in the culture medium; whereas at 100 mg L -1 , microbial manifestation decreased in 65% of the explants of individual 2.
Streptomycin at the concentration of 100 mg L -1 has been considered e cient to control microbial manifestation in other in vitro plant cultures: in Musa spp. (banana tree), it reduced microbial manifestation [48]; in Solanum tuberosum (potato), it inhibited the growth of bacterial manifestation [34]; and in Rauwol a serpentina, it completely removed apex shoot microbial manifestations [35]. These reports align with our ndings for in vitro cultures of Eucalyptus microcorys, whereby streptomycin at 100 mg L -1 showed the best result by eliminating microbial manifestations in 65% of all explants of individual 2.
In the genus Aglaonema, there is a direct relationship between streptomycin concentration and microbial reduction [49]. Increasing the concentration of streptomycin from 25 to 400 mg L -1 decreased microbial manifestations percentages from 83.3% to 0%. Here, we found an inverse relationship between these variables: increasing concentrations of streptomycin were followed by decreasing e ciency in terms of reduction of microorganism manifestation.
Regarding sodium hypochlorite, several studies have reported its effects on the reduction of microbial manifestation in culture media. At low active chlorine concentrations (up to 0.005%), sodium hypochlorite has proven viable in controlling bacterial manifestation in in vitro cultures of Eucalyptus benthamii at the establishment phase [16], Eucalyptus pellita at the multiplication phase [22], Sequoia sempervirens L. [21], the hybrid Gerbera hybrida cv. Essandre [24], Cochlospermum regium [25] and Bambusa vulgaris [26]. Our results for Eucalyptus mycrocoris agree with the literature: sodium hypochlorite at 0.003% produced the best response by eliminating microbial manifestations in 25% of individual 2 explants and 5% of individual 1 explants. The addition of chemical agents to in vitro cultures is a viable alternative to avoid culture loss and discard. But it is essential to choose a suitable concentration and carefully handle these products to avoid phytotoxicity to the plants and achieve success [50], considering the possibility that the microorganisms build up resistance against these chemical agents [31]. Our results pointed to sodium hypochlorite as a consistent option for its low cost, for its e ciency and for lacking phytotoxic effects on in vitro cultures of Eucalyptus microcorys. The antibiotic streptomycin, at 100 mg L -1 , was also e cient as it was able to reduce microbial manifestation without interfering in the viability or survival of explants.

Conclusion
The addition of sodium hypochlorite and streptomycin to the culture media was e cient for the survival of explants and for the reduction in microbial manifestation in in vitro cultures of Eucalyptus microcorys. Individuals 1 and 2 displayed similar responses in terms of survival. Regarding reductions in microbial manifestation, explants of individual 2 displayed a better response to the chemical agents than individual 1.
The best result was achieved with the addition of streptomycin at 100 mg L − 1 to the culture medium, which led to 95% of explant survival and 65% of reduction in microbial manifestation in individual 2.
The addition of sodium hypochlorite at 0.003% to the culture medium resulted in an explant survival percentage of 100%, but was less e cient than streptomycin, which eliminated microbial manifestation in 25% of the explants of individual 2. Illustration of the experimental methodology employed to reduce microbial manifestation in Eucalyptus microcorys explants in vitro cultivated