Rhizobial inoculation in black wattle plantation (Acacia mearnsii De Wild.) in production systems of southern Brazil

Abstract

Black wattle (Acacia mearnsii De Wild.) is a tree legume native to southeast Australia, but present in all continents. Today it covers about 142,400 ha in Brazil, with plantations concentrated in the southern region of the country. Black wattle may form nodules and establish rhizobial symbiosis capable of fixing N₂, but rhizobial inoculation is not done in commercial plantations. About 40 kg ha⁻¹ of urea is applied during seedling transplantation. In this review, evidences by which rhizobial inoculation affects monoculture, mixed cultivation, and agroforestry black wattle production systems were searched in literature. Previous measurements in cultivated forests have indicated that biological nitrogen fixation in black wattle may provide up to 200 kg of N ha⁻¹ year⁻¹ to the soil. Therefore, rhizobia inoculation may bring several opportunities to improve black wattle production systems. Black wattle is not a very selective partner in the rhizobial symbiosis, but the genus Bradyrhizobium dominates the rhizobial diversity of black wattle nodules. Investigation on rhizobial diversity in soils where the crop is cultivated may represent an opportunity to find more effective rhizobia strains for inoculants. The successful history of biological nitrogen fixation in grain legumes must inspire the history of tree legumes. Microbiology applied to forestry must overcome challenges on the lack of trained professionals and the development of new application technologies.

Introduction

Acacia mearnsii De Wild. (black wattle) is a tree legume native to southeast Australia, but present in all continents. It is the fourth most planted tree species in Brazil [1], with the first plantation reported at the beginning of the twentieth century for the production of tannin, used in the leather industry. In 2016, Brazil produced 230,000 tons of black wattle bark for tannin extraction, worth about 49.6 million dollars in exports to India, Mexico, and China [2]. Cropping of black wattle in Brazil started with 30 kg of seeds imported from South Africa to Southern Brazil (State of Rio Grande do Sul) by the company SETA S.A. [3]. Today it is mainly planted in the Southern Brazil, where climate favors the crop, in 142,400 ha, administrated by two companies: SETA S.A. and TANAC S.A. [2]. Indeed, the actual area covered with black wattle may be much larger as cultivation by smallholding farmers is not precisely measured [4] since the estimated number of smallholding family farmers growing black wattle has increased from 4400 in 1980 [5] to 35,000 in 2015 [4]. The production of black wattle in smallholding family farms is mostly possible by integration of objectives of smallholding farmers and the tannin extraction industry. In general, the integration is good for all stakeholders involved. On the one hand, industry receives a steady flow of raw material and can better control the quality of it; on the other hand, the smallholding farmer is assured with income. For those reasons, management of black wattle production has been considered to be the determining factor for the sustainability of the agroecosystems in Southern Brazil, reducing emigration from rural areas and erosion rate [6].

In South Africa, black wattle has endangered biodiversity of natural reserves because despite being an important crop, it became an aggressive invasive alien species. In Brazil, black wattle (cropped mainly in State of Rio Grande do Sul) has been encountered in degraded native ecosystems and marginal areas [7] in the States of Santa Catarina, Paraná, and Rio de Janeiro e Paraíba (approximately 3900 km far away) but did not affect the ecosystems functioning [8].

Although black wattle was first introduced to Brazil to produce tannin, nowadays it is cultivated for multiple uses. Black wattle bark is raw material for leather tanning, adhesives, resins, chelates, and preservatives [9–11]. Its timber is a source of fibers (cellulose), wood, wood pellets, and charcoal [11, 12]. Furthermore, after harvest, residues of roots and aerial parts add carbon, nitrogen, and other nutrients to the soil, which through soil organic decomposition process improves soil nutrient cycling and soil fertility [6, 13].

Being a legume, black wattle can establish a symbiotic association with diazotrophic bacteria, commonly known as rhizobia. Rhizobia comprise several bacteria genera that stimulate nodule formation in legume and reduce N₂ into NH₃ in exchange of photosynthate. Biological nitrogen fixation (BNF) is a process that allows nitrogen (N) to enter the system, so that N is available in the soil, benefiting plants, animals, and soil microbial biomass [14]. Symbiosis between legumes and rhizobia accounts for about 20–25% of the income of N into the N cycle of an ecosystem [15].

Measurements in cultivated forests more than 30 years old of South Africa have indicated that BNF may provide up to 200 kg of N ha⁻¹ year⁻¹ in black wattle to the soil [16]. However, efficiencies in the process vary from 10 to 100% (Table 1), according to the environment and plant age. For example, it was found that nodules were more efficient in black wattle of the first cycle and less efficient in second and the third cycles of production [20]. Interestingly, even away from its original ecosystem, black wattle may form symbiotic nodules with strains of rhizobia that are present in its novel environment (e.g., the authors Pedro Henrique Riboldi Monteiro and Celso Garcia Auer have often encountered root nodules in black wattle in the plantations in Brazil). However, it is unclear whether nodulation with these non-co-evolved rhizobia would provide symbiotic performance similar to co-evolved rhizobia (i.e., high rates of N₂ fixation). Isolation of adapted rhizobia from field growing nodules may be an opportunity to find more efficient strains [17].

Table 1.

Productivity and biological nitrogen fixation in black wattle in different parts of the world

System local	Age (years)	Timber productivity (m3 ha−1 year−1)	Rates of FBN (Kg N ha−1 year−1)	Method of FBN estimation	Reference
Monoculture
Australia	10	5.4	86.2	N addition	[17]
			131.5 (63%)	15N
South Africa	10–12	5–19			[11]
Brazil	6–8	13–26
South Africa	30		200	N difference	[16]
Mixed system
Australia	10	7.8	38.3	N addition	[17]
			16.2 (10%)	15N
Invasive forest
South Africa			(75%)	15N	[18]
Native forest
Australia			0.75	Acetylene reduction	[19]

In this review, it was investigated whether rhizobial inoculation would increase black wattle productivity and ensure crop sustainability through improved BNF.

Production systems for black wattle with emphasis in Brazil

Commercial fields of black wattle are usually planted with 15 to 30-cm seedlings in Southern Brazil that take about 4 years to start flowering and fruiting and 7 years to reach harvest heights, that is, heights of up to 25 m. The crop may be planted as monoculture, combined with other forest species (mixed planting systems), or in combination with annual crops such as common beans, maize, vegetables, others, and/or with pastures (agroforestry systems). In the case of Brazil, the majority of black wattle plantations are managed under monoculture (e.g., 70–85%) and the rest are managed in mixed or agroforestry systems (mainly in smallholding farms).

Table 2 depicts a number of advantages and disadvantages of the three main systems in Brazil. These advantages and disadvantages depend on the complexity of the systems, resulting from the sum and the interaction of the species that compose them [33, 34]; on the available technologies; and on trained labor (particularly in the case of agroforestry) [35, 36].

Table 2.

Advantages and disadvantages of the production systems for black wattle, with emphasis in south Brazil*

Advantages	Disadvantages
Monoculture
Stand uniformity/one species at a time Well-developed technology available Easy access to credit lines More information available	Worse land use Long-term financial return Not accessible to smallholding farms High dependence on external resources Increased use of agrochemical products High export of nutrients from the soil
Mixed planting
More productive than monoculture Higher rates of nutrient cycling More balanced use of nutrients in the system Greater biological diversity Optimization of soil use by species of different root systems More sources of income for the farm	Not suitable for large scale production More demanding on management and interventions Non-immediate income return Higher implantation cost per area Lower access to credit line programs Lack of information or technical support
Agroforestry
Higher soil and water quality Increased biodiversity Greater diversification in production Maintenance and retention of organic matter and nutrient cycling Good animal comfort and welfare More sources of income (short, medium, long term) Amortization of production costs Decreased dependence on external fertilizers Nutritional exploitation of soil in lateral extension and depth Nutritional enrichment of soil Optimization of soil use by species of different root systems Better condition of soil physical properties Animal feed supplementation Higher N use efficiency of the system Higher productivity Reduction of the risk of productivity loss Food security and self-sufficiency Conservation of natural genetic resources	Not suitable for large scale production of timber Requires trained labor force Greater number of management stages and cultural practices (silvicultural, agricultural, pasture, and livestock) Higher implantation cost/ha Shortage of technologies Few credit line programs Lack of information or technical support

Based on [5, 11, 21–32]

Monoculture

The monoculture consists of a system of land use in which only a single crop is cultivated. This system is usually associated with large-scale production driven by the high demand of raw material (e.g., wood and extracts). Expansion of monoculture black wattle plantations has been a result of the development in plant genetic breeding, improved soil fertility management, silvicultural methods, irrigation, and mechanized harvesting. The main advantage of monoculture is that trees grow with a more uniform architecture, which makes processing easier and improves the yield rates through intensive land use [21, 37, 38].

However, monoculture requires higher levels of soil nutrients (e.g., 40 kg ha⁻¹ of urea and 80 kg of P₂O₅ ha⁻¹ during the implantation of the forest [39]) and consumes more energy coming from fossil fuels through the production of chemical fertilizers. The use of industrial N fertilizers interferes directly in the N cycle because it is responsible for about 40% of the entire N circulating on the planet [15]. In addition to monetary costs, excess N supplied by fertilizers may cause environmental pollution through leaching and volatilization [40]. Furthermore, conventional agricultural production based on the applications of industrial fertilizers affects species, diversity, dynamics, and functionality of agroecosystems [41].

Furthermore, to be sustainable, an activity must be economically viable, socially just, and environmentally sound [42]. The facts mentioned here make the conventional model of black wattle production quite unsustainable. Thus, monoculture production of black wattle may negatively affect its sustainability. However, a shift of total or partial chemical fertilization to natural BNF would improve the sustainability of agriculture [43] because once symbiosis is established with efficient strains, plants have better N nutrition and, therefore, contribute to increasing the level of N cycling in the plant-soil system. As the crop deposits a substantial amount of litter and fresh residues, it promotes soil microbial biomass and nutrient cycling.

Mixed system

The mixed planting system consists of cultivating two or more forest species under consortium, at the same time on the same land area. The success of this production system relies on the arrangement of plant components that decreases competition among them [44]. Mixed systems are very dynamic as they change in the scale that their components grow and develop, and they respond to the relationships among the species involved and to the availability of resources [22]. Therefore, mixed systems tend to be more productive than monoculture systems. To date, a meta-analysis considering the overall response of forest biomass indicated that mixed systems produced 24% more biomass than monoculture [45]. The factors that affect species productivity in mixed systems are stocking density and age, resource availability, climatic conditions, and environmental disturbances [45]. Furthermore, the success of mixed planting systems is determined by nutrients cycling and decomposition of soil organic matter, which is built up from deposition of plant biomass (particularly roots) and release of organic compounds by roots to the soil [6, 46, 47].

One example of forest mixed systems is the one of black wattle with Eucalyptus in Brazil. Field experiments have shown that the mixed system of black wattle with Eucalyptus optimizes the use of soil resources, since the crops have distinct root systems and exploit different soil niches [23]. In addition, Eucalyptus grows rapidly and requires a greater amount of sunlight than black wattle, which naturally occurs as an understorey species and is more shade tolerant [48, 49]. For example, in a black wattle-Eucalyptus globulus 10-year-old plantation in Australia, both forest species grew faster in the mixed system than in monoculture [46].

Inclusion of legumes in Eucalyptus plantations may increase the capacity of soil nutrient recycling. For example, in an experiment in Rio de Janeiro, Southeastern Brazil, after 30 months of plantation, Eucalyptus urograndis with Acacia mangium resulted in more soil nutrient available than the monoculture of E. urograndis [50]. The mixed systems were probably benefited from the A. mangium-rhizobia symbiosis, which has the potential of increasing N levels in the litter, dead roots, and plant exudates [50].

However, mixed and agroforestry system productivity should not be evaluated individually but as a whole. If the production of each agroforestry component or a mixed composition is evaluated alone, productivity will seem not feasible since increased plant diversity may not increase the productivity of specific tree species [45]. Nevertheless, mixed plantations imply more sources of income through assorted rotations resulting in economic satisfaction for small and medium holding farms, if they do not depend on immediate returns. Furthermore, when management with rhizobia is well applied, there are ecological benefits as it increases the capacity of soil nutrient cycling, which may decrease the agricultural inputs in the system.

Agroforestry systems

The agroforestry system consists of forestry plantation in consortium with annual crops, and occasionally pastures, which feed livestock cattle. As forestry systems (monoculture or mixed systems) do not produce rapid financial return, the agroforestry systems can be a viable alternative to obtain an extra income (with livestock production, for example) within a shorter period of time, prior to logging, reducing the overall production costs of forest products. In addition, the inclusion of forest in livestock and agricultural production systems promotes biodiversity, stimulates nutrient cycling, increases farm profits, and decreases dependence on external inputs [51, 52]. Therefore, agroforestry is thought to be the most sustainable production system integrating forest, agricultural, and livestock components at the same time in the same area [34, 53]. Agroforestry systems may have a greater return than monoculture systems due to the diversified component production in consortium [5, 54–56]. Examples of the benefits coming from this system are higher yields of timber and wood and greater animal weight gain due to greater feed availability and animal thermal comfort [51, 57].

Smallholding farmers from Southern Brazil have implemented agroforestry systems that include black wattle and Eucalyptus, intercropped with cassava (Manihot esculenta), common beans (Phaseolus vulgaris), maize (Zea mays), potato (Solanum tuberosum), and watermelon (Citrullus lanatus), among others. In the agroforestry system of Southern Brazil, the animals are usually brought to graze when black wattle trees reach 7 to 10 m in height because annual crops can no longer develop due to shading caused by trees [34]. In that case, for the agroforestry to function well, the black wattle planting density has to be adjusted. For example, growth of black wattle and different pastures were not affected by black wattle different planting densities (500 and 833 trees ha⁻¹) up to the fifth year [51]; however, the yield of pastures decreased under high black wattle planting densities (833 plants ha⁻¹) from the fifth year onwards [34]. Trees and annual crops have different strategies to take up nutrients, by their own roots or by including the choice for mycorrhizal fungi species that facilitate the absorption of different nutrients [58, 59]; meanwhile, legume adds N through the symbiosis with rhizobia [24, 60]. Furthermore, in addition to the positive effect of soil organic matter improvement, it is possible that rhizobia inoculation upon black wattle under agroforestry could result in forage of better quality, which in turn, could have benefits on plant growth and indirect effects on livestock weight gains [51].

Strengths, weaknesses, threats, and opportunities

Papers that dealt with the black wattle production systems (monoculture, mixed planting, and agroforestry systems) in any part of the world under two scenarios—with and without effective BNF (promoted by rhizobial inoculation) in black wattle—were considered for this review. The review revealed that inoculation of black wattle with rhizobia promotes plant growth, productivity, and sustainability in monoculture, mixed, and agroforestry production systems. Table 3 highlights that rhizobia inoculation results in more points of strengths than points of weaknesses. It implies that rhizobia inoculation may be regarded as a silvicultural practice that can be controlled, bringing direct results to the management.

Table 3.

Strengths, weaknesses, threats, and opportunities of rhizobia inoculation in production systems of black wattle

Strengths ✓ Ensuring the establishment of symbiosis ✓ Increased plant growth and yield ✓ Improved environmental quality ✓ Improved plant nutrition ✓ Stimulation of soil nutrient cycling ✓ Decreased dependence of N fertilizers	Weakness ✓ Uncertainties in knowledge about efficiencies in the association between rhizobia and forest species ✓ Development of inoculation techniques ✓ Lack of expertise in microbiology in the forest sector
Opportunities ✓ Greater technical and scientific development ✓ Development of the forest ✓ Microbiology in the forestry sector ✓ Product certification for using biotechnological products based on rhizobia	Threats ✓ Need investment for research and technological development ✓ Unfair market competition between biotechnology and conventional fertilizers

One of the weaknesses of the rhizobial inoculation is the uncertainty in knowledge about specific efficiency of rhizobial diversity. In soils with low rhizobium density, the establishment of symbiosis and the occurrence of BNF are uncertain [61, 62]. The application of inoculants with pre-selected rhizobia should ensure an efficient root infection and nodule formation (Table 3) and, consequently, increase plant growth and productivity. Therefore, the success of rhizobia inoculation as a silvicultural practice depends on the choice of rhizobia strains that perform well in the symbiotic association and have the highest potential for BNF. In addition, these strains must cope with competition with native strains, as they may have better infectivity properties than inoculated strains [63].

Once rhizobial inoculation ensures establishment of symbiosis, crops are benefited from enhanced nutrition and stimulated photosynthesis and growth [48, 64, 65], promoting soil fertility by increasing the amount of plant residues with lower C to N ratio [46]. The soil organic matter with lower C to N ratios can benefit soil microbial biomass and increase soil nutrient cycling capacity [46]. Rhizobia inoculation can improve soil quality and increase land use efficiency. Furthermore, the adoption of rhizobial inoculation in black wattle systems brings great opportunities such as (1) greater technical and scientific development of the crop, (2) development of forest microbiology in the forest sector, and (3) certification of products for the use of biotechnology products based on rhizobia.

If rhizobia inoculation is a good thing, why is it not done?

Legume promiscuity, nodule effectiveness, and rhizobia competitiveness

Legumes have evolved to permit nodulation with different strains within a spectrum of species to guarantee nodulation under oscillating and severe stressful environments [66]. The majority of tropical legumes have a “promiscuous” nature and do not restrict nodulation to one specific rhizobia strain [66–68]. In other words, one could not expect that black wattle would naturally form nodules with only one type of rhizobia. In addition, rhizobia diversity includes variability to expression of different molecules (nif and nod factors, for example) that stimulate or depress nodule activity, increasing or decreasing nodule effectiveness [63]. If legumes preferred specific strains, that is, the ones with higher rates of BNF, then inoculation would be overpowering. But, due to legume promiscuity and the higher competitiveness of native strains in relation to exotic strains, seedling inoculation would be advantageous because it places the rhizobia cells next to the developing rooting system at an early stage [63, 69].

Studies of rhizobial diversity in nodules of black wattle planted in Australia and South Africa have identified strains belonging to species Bradyrhizobium japonicum, B. elkanii, Rhizobium leguminosarum, and R. tropici [70]. But, the genus Bradyrhizobium is considered to be the dominant symbiotic rhizobia in black wattle nodules [17, 40, 69, 71–73]. To date, the genus Bradyrhizobium represented 97% of the strains isolated from black wattle growing in Australia [72], 100% of the strains isolated from nodules of commercial black wattle plantations in Brazil [17], and 100% of strains isolated from nodules growing in alien and native forests in Algeria [40].

Therefore, considering their abundance in black wattle plantations, Bradyrhizobium are probably the most efficient strains for BNF symbiosis. Indeed, inoculation of three species of Bradyrhizobium in black wattle resulted in more vigorous seedlings than the counterpart non-inoculated seedlings [73]. When these same inoculated and vigorous seedlings were transplanted to the field in an experimental area of Australia under monoculture or mixed planting systems in consortium with Eucalyptus nitens (Deane and Maiden) Maiden, they resulted in larger trees in both the black wattle and in eucalyptus [73].

In Brazil, the Normative Instruction 13/2011 of the Ministry of Agriculture, Livestock, and Supply [74] has authorized two strains, Bradyrhizobium japonicum (SEMIA 6163 = AY904764 = BR 3607) and B. japonicum (SEMIA 6164 = AY904765 = BR 3608), isolated in Seropédica, Rio de Janeiro, Brazil, to be used in inoculants for black wattle. Authorized strains are available from the Brazilian Rhizobium Culture Collection SEMIA at the Brazilian Agricultural Research Corporation (EMBRAPA-Soja, Londrina, Paraná, Brazil) and FEPAGRO-MIRCEN (Fundação Estadual de Pesquisa Agropecuária (Rio Grande do Sul, Brazil) – Microbiological Resources Center) (IBP World Catalogue of Rhizobium Collections no. 443 in the World Federation of Culture) [75]. Official regulations consider that only strains with outstanding results of plant growth promotion are authorized for inoculants. However, despite being accessible for public use, authorized strains for black wattle and other forest legume species are not available in the market as inoculants. The reason is that inoculant industry would rather prefer to have only one strain for all seedling species than one rhizobium strain to each species as currently proposed by the Brazilian Normative Instruction. Furthermore, the commercial forestry production systems are not used to inoculation as the annual cropping systems are.

With the understanding that rhizobia-legume symbiosis is governed by legume promiscuity and competition among soil rhizobia strains, investigation on rhizobial diversity in soils where the crop is cultivated may represent an opportunity to find more effective rhizobia strains for crops and forest species [17, 40]. The successful history of BNF in grain legumes must inspire the history of tree legumes. In the case of soybean, BNF is enough to supply all N demands of the crop without the need of N fertilization [76].

Efficient means for application of inoculants

Commercial inoculants are made available by the inoculant industry in solid (peat) and (semi-)liquid formulations [77]. Peat is the best carrier because it preserves microorganism cells, being used in both solid and semiliquid formulations. Regardless of formulations, inoculants for grain legumes, and particularly for soybean, dominate the market, with over 50 million doses (1 dose per hectare) sold in 2016 [77] whereas forest species are hardly inoculated.

In fact, although several institutes have researched the diversity of rhizobia of tree legumes [17] and different techniques for inoculating microorganisms in seedlings [78, 79], few investigations resulted in commercial inoculants for forest species [68]. Some inoculants for forest species are made available in the market as “organic fertilizer” and, when they are used under nursery seedlings or transplantation, e.g., [79], they are not treated as an alive product as they should [78]. Therefore, research is challenged to improve knowledge about inoculation technology as a whole. Considering the initial slower growth rates of forest species compared with the growth rates of annual crops, forest inoculants must provide stronger conditions for survival of microorganisms than the inoculants for annual crops species provide. A possible alternative to be developed would be having inoculant carriers that preserve microbial cells for longer time and release them slowly as the crop seedling develop. Moreover, regarding inoculation timing, a question to be answered is whether it would be more efficient to apply inoculants at time of seedling, for example, on seeds or in substrate, or during transplantation, for example in the holes of planting, and, even so, after transplantation in a topdressing way. These are technological aspects that have not been addressed in literature concerned with black wattle and other Acacia species.

Forest microbiology

Our paper summarized that BNF is not fully exploited in black wattle production systems in Brazil, although it is a promising technology to increase plant growth, yields, and crop sustainability. Therefore, there is an urge for promoting projects and programs in the theme. In addition to rhizobia inoculants, the situation is extended to other microorganisms that promote plant growth, support biological control, phosphate solubilization, and mycorrhizal fungi, among others.

Even though the forestry sector has a large scale in the country as well as in the world, it focuses only on a few species. Microbiology applied to forestry must overcome some challenges, such as the lack of trained professionals to develop research, as well as the development of new application technologies. It could bring greater opportunities for scientific and technological research through the production of new inoculants, in a new way and with higher inoculation efficiency.

Concluding remarks

The greatest threat to the development of inoculants in the forestry microbiology is the need for high investments in research. Another threat is the unfair way that market of biological and synthetic fertilizers works. Fertilizers have well-established industrial settlements and dominate the market when compared with biological products.

Overall, the present work raised up the need to develop forestry microbiology. The biotechnological innovations resulting from the results of the research on microbial diversity and ecology will bring new opportunities in silvicultural practices and forest production.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.