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Do plants have preference for the form of nitrogen as nutrient?

Do plants have preference for the form of nitrogen as nutrient?


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In the nitrogen cycle (ecology), it is usually described that plants can use nitrogen in the form of ammonium (NH4+) and nitrate (NO3-). Do plants prefer one form of nitrogen over the other?


Quoting verbatim from this site. The reference is not really a scientific article but you can check the references it cites. Some were not in English so I did not check. However these points are fairly logical

Nitrates are the preferred nitrogen source:

  • Non-volatile: unlike ammonium, nitrate is non-volatile, so there is no need to incorporate it in the soil when applied by top- or side dressing, which makes it a convenient source for application.

  • Mobile in the soil - direct uptake by the plant, highest efficiency.

  • Nitrates synergistically promote the uptake of cations, such as K, Ca and Mg, while ammonium competes for the uptake with these cations.
  • Nitrates can be readily absorbed by the plant and do not need to undergo any further conversion, as is the case with urea and ammonium, before plant uptake.
  • No acidification of the soil if all the nitrogen is applied as nitrate-nitrogen.
  • Nitrates limit the uptake of harmful elements, such as chloride, into large quantities.
  • The conversion of nitrates to amino acids occurs in the leaf. This process is fuelled by solar energy, which makes it an energy-efficient process. Ammonium has to be converted into organic N compounds in the roots. This process is fuelled by carbohydrates, which are at the expense of other plant life processes, such as plant growth and fruit fill.

Many plants can carry out nitrification but this is not universal. From this article:

Nitrification is sometimes considered so universal and rapid that applications of NH4-N are considered equivalent to NO3-N. This is not true in many forest, orchard, and grassland soils.

However, nitrate being more soluble is susceptible to leaching and wash off, care has to be taken when using nitrate based fertilizers. From the previously mentioned paper:

Unlike the positively charged ammonium ion, which is relatively stationary because of its adsorption to organic matter or clay particles, the negatively charged nitrate ion is freely mobile in the soil solution (166). Thus, leaching and denitrification primarily involve a loss of NO3-N (171). Inhibition or retardation of nitrification of applied NH4-N can reduce nitrogen losses, increase efficiency of applied N, and establish a predominantly ammoniacal form of nitrogen available for plant uptake (105, 171,222, 232).


Secondary Plant Nutrients: Calcium, Magnesium, and Sulfur

Calcium, magnesium, and sulfur are essential plant nutrients. They are called “secondary” nutrients because plants require them in smaller quantities than nitrogen, phosphorus, and potassium. On the other hand, plants require these nutrients in larger quantities than the “micronutrients” such as boron and molybdenum.

Calcium, magnesium, and sulfur are generally adequate in most Mississippi soils with favorable pH and organic matter levels. They affect pH when applied to the soil. Calcium and magnesium both increase soil pH, but sulfur from some sources reduces soil pH. Compounds containing one or more of these nutrients are often used as soil amendments rather than strictly as suppliers of plant nutrition.

Calcium

The primary function of calcium in plant growth is to provide structural support to cell walls. Calcium also serves as a secondary messenger when plants are physically or biochemically stressed.

Calcium deficiencies rarely occur in Mississippi soils. Soils with favorable pH levels are normally not deficient in calcium. Acid soils with calcium contents of 500 pounds per acre or less are deficient for legumes, especially peanuts, alfalfa, clovers, and soybeans. At this level, limited root system crops such as tomatoes, peppers, and cucurbite would also need additional calcium. Soluble calcium is available as the Ca2+ ion and is needed for peanuts at pegging time and for peppers and tomatoes to prevent blossom end rot.

Available calcium can be lost from the soil when it is (a) dissolved and removed in drainage water, (b) removed by plants, (c) absorbed by soil organisms, (d) leached from the soil in rain water, or (e) absorbed by clay particles. Deficiency symptoms include death at the growing point, abnormally dark green foliage, weakened stems, shedding flowers, and any combination of these.

Limestone is the primary source of calcium in Mississippi. Other common sources include basic slag, gypsum, hydrated lime, and burned lime. These sources are recommended for peanuts, peppers, and tomatoes. In peanuts, they prevent pops and encourage pegging. In tomatoes and peppers, they prevent pops and blossom end rot. Hydrated lime and burned lime contain more readily available calcium than do basic slag and gypsum. Gypsum does not affect soil pH even though it contains calcium.

Magnesium

Magnesium is adequate for crop production in most Mississippi soils except the coarse sandy soils of the Coastal Plains and the heavy dark clays of the Blackbelt Prairie. Magnesium is absorbed as the Mg2+ ion and is mobile in plants, moving from the older to the younger leaves. It leaches from the soil like calcium and potassium.

Magnesium is the central atom amid four nitrogen atoms in the chlorophyll molecule, so it is involved in photosynthesis. It serves as an activator for many enzymes required in plant growth processes and stabilizes the nucleic acids.

Interveinal chlorosis is a deficiency symptom in crops such as legumes, corn, sorghum, cotton, and certain leafy vegetables. (Interveinal chlorosis is a yellowing between the veins while the veins remain green.) The leaves may become pink to light red and may curl upward along the margins.

To correct magnesium deficiency in soil, use dolomitic lime when lime is needed use soluble sources of magnesium when lime is not needed. Magnesium supplementation may be needed for cotton production in the Blackland Prairie.

Cattle are often affected by grass tetany when forage magnesium content is low. Other factors include nitrogen, calcium, and potassium levels, stage of growth (usually in spring), whether or not cattle are lactating, and seasonal conditions. Dolomitic limestone is recommended as a liming material where grass tetany has been a problem. Give grazing animals supplemental magnesium and calcium when grass tetany is an issue. For more detailed information on grass tetany issues, see Extension Publication 2484 Mineral and Vitamin Nutrition for Beef Cattle.

The most common soluble sources of magnesium to use as fertilizer are magnesium sulfate (containing 10% Mg and 14% S, also known as Epsom salt), sulphate of potash magnesia (containing 11.2% Mg, 22% S, and 22% K2O, commercially sold as K-Mag), and magnesium oxide (containing 55% Mg, also known as magnesia).

Sulfur

Sulfur is needed in fairly large quantities by most crops. It is an essential building block in chlorophyll development and protein synthesis. Sulfur is required by the rhizobia bacteria in legumes for nitrogen fixation. In general, crops remove about as much sulfur as they do phosphorus. Grasses remove sulfur more efficiently than legumes, and clovers often disappear from pasture mixtures when sulfur is low.

The sulfate ion, SO4, is the form primarily absorbed by plants. Sulfate is soluble and is easily lost from soils by leaching. As sulfate is leached down into soil, it accumulates in heavier (higher clay content) subsoils. For this reason, testing for sulfur in topsoil is unreliable for predicting sulfur availability during a long growing season.

Many coarse-textured, sandy soils and loworganic matter, silty soils throughout Mississippi are sulfur deficient for crop production. Many acid soils contain metallic sulfides that release sulfur as weathering occurs.

Sulfur deficiency symptoms show on young leaves first. The leaves appear pale green to yellow. The plants are spindly and small with retarded growth and delayed fruiting. For a rapid correction of a deficiency, use one of the readily available sulfate sources.

Sulfur may be recommended for major crops in Mississippi at 8–10 pounds per acre annually in some situations. Check with local MSU Extension Service offices or area agronomists for more crop- and sitespecific information.

There are many sources of fertilizer sulfur available. Organic matter is the source of organic sulfur compounds and is the main source of soil sulfur in most Mississippi soils. Other sources of sulfur are rainfall and fertilizers that contain sulfur. Some readily available sources include ammonium sulfate (21% N and 24% S), potassium sulfate (50% K20 and 17.6% S), gypsum (32.6% CaO and 16.8% S), and zinc sulfate (36.4% Zn and 17.8% S).There are several other sulfate sources as well as less available sources of sulfur in the elemental or sulfide form.

Elemental sulfur is a good acidifying agent. An application of 500 pounds of sulfur per acre on sandy loam soil reduces the pH from 7.5 to 6.5. It takes about 3 pounds of lime to neutralize the acidity formed by 1 pound of sulfur.

Table 1. Average percentage of chemical content of major sources of calcium magnesium, and sulfur.


The Chemical Composition of Plants

Since plants require nutrients in the form of elements such as carbon and potassium, it is important to understand the chemical composition of plants. The majority of volume in a plant cell is water it typically comprises 80 to 90 percent of the plant&rsquos total weight. Soil is the water source for land plants, and can be an abundant source of water, even if it appears dry. Plant roots absorb water from the soil through root hairs and transport it up to the leaves through the xylem. As water vapor is lost from the leaves, the process of transpiration and the polarity of water molecules (which enables them to form hydrogen bonds) draws more water from the roots up through the plant to the leaves (Figure (PageIndex<1>)). Plants need water to support cell structure, for metabolic functions, to carry nutrients, and for photosynthesis.

Figure (PageIndex<1>): Water is absorbed through the root hairs and moves up the xylem to the leaves.

Plant cells need essential substances, collectively called nutrients, to sustain life. Plant nutrients may be composed of either organic or inorganic compounds. An organic compound is a chemical compound that contains carbon, such as carbon dioxide obtained from the atmosphere. Carbon that was obtained from atmospheric CO2 composes the majority of the dry mass within most plants. An inorganic compound does not contain carbon and is not part of, or produced by, a living organism. Inorganic substances, which form the majority of the soil solution, are commonly called minerals: those required by plants include nitrogen (N) and potassium (K) for structure and regulation.


Forms of essential plant nutrients

To be used by a plant, an essential nutrient must be broken down into its basic form. The nutrient must be in the form of either a positively charged ion (cation) or a negatively charged ion (anion). A plant cannot use organic compounds, such as those in manure or dead leaves, until they are broken down into their elemental or ionic forms.

Also, plants cannot use an element that is not in the proper form (a specific ion) even if it is present in high concentrations in the soil. For example, the presence of iron (Fe) in the soil will not guarantee that enough of the proper iron ions, Fe2+ or Fe3+, will be available to the plant.

Plants take in almost all of the essential nutrients through their roots. The exception is carbon, which is taken in through leaf pores, or stomata. Two types of organisms living in the soil help the roots take up nutrients:

  • Microorganisms, or microbes, break down organic compounds into inorganic compounds in a process called mineralization.
  • Fungi enable some plants to take up phosphorus by increasing the size of the roots and providing more soil-to-root contact.

Different inter-annual responses to availability and form of nitrogen explain species coexistence in an alpine meadow community after release from grazing

Plant species and functional groups in nitrogen (N) limited communities may coexist through strong eco-physiological niche differentiation, leading to idiosyncratic responses to multiple nutrition and disturbance regimes. Very little is known about how such responses depend on the availability of N in different chemical forms. Here we hypothesize that idiosyncratic year-to-year responses of plant functional groups to availability and form of nitrogen explain species coexistence in an alpine meadow community after release from grazing. We conducted a 6 year N addition experiment in an alpine meadow on the Tibetan Plateau released from grazing by livestock. The experimental design featured three N forms (ammonium, nitrate, and ammonium nitrate), crossed with three levels of N supply rates (0.375, 1.500 and 7.500 g N m −2 yr −1 ), with unfertilized treatments without and with light grazing as controls. All treatments showed increasing productivity and decreasing species richness after cessation of grazing and these responses were stronger at higher N rates. Although N forms did not affect aboveground biomass at community level, different functional groups did show different responses to N chemical form and supply rate and these responses varied from year to year. In support of our hypothesis, these idiosyncratic responses seemed to enable a substantial diversity and biomass of sedges, forbs, and legumes to still coexist with the increasingly productive grasses in the absence of grazing, at least at low and intermediate N availability regimes. This study provides direct field-based evidence in support of the hypothesis that idiosyncratic and annually varying responses to both N quantity and quality may be a key driver of community structure and species coexistence. This finding has important implications for the diversity and functioning of other ecosystems with spatial and temporal variation in available N quantity and quality as related to changing atmospheric N deposition, land-use, and climate-induced soil warming.


Acknowledgements

We thank Jim Dalling, Gregory Goldsmith, Nelly Ramos, Didimo Ureña, David Navarro and Leidys Rodriguez for their assistance with fieldwork and plant preparation and Dayana Agudo and Tania Romero for laboratory support. Funding was provided by the University of Illinois - Champaign/Urbana Programme in Ecology, Evolution, and Conservation Biology Summer Research Award and the Govindjee and Rajni Govindjee Award and STRI Postdoctoral Fellowship to KMA. The Smithsonian Tropical Research Institute and Enel Fortuna provided logistical support. Modified needles were provided by Jan Jansa and Fabienne Zeugin in the Plant Nutrition Group at ETH Zurich. Jim Dalling, Jordan Mayor and two anonymous reviewers provided helpful comments on previous versions of the manuscript.


Anions and cations in plants, oh my! But why do we care?

Plant essential nutrients exist as anions and cations. What does that mean for plant production and why should growers be aware?

There is a lot of biological and chemical activity occurring in your average soil. Most happens unseen and unheard, and that&rsquos probably a good thing. Just because these organisms and processes don&rsquot scream out, doesn&rsquot mean growers shouldn&rsquot pay attention to what goes on. In fact, paying attention can make the difference between a good and a great grower. In an earlier Michigan State University Extension article, &ldquoKnowing nutrient mobility is helpful in diagnosing plant nutrient deficiencies,&rdquo I discussed nutrient mobility within the plant and how understanding mobility helps identify nutrient deficiencies, but it is also important for growers to pay attention to nutrient mobility in the soil.

Plant nutrients exist in the soil as either anions or cations. What are they? Most molecules in natural systems have a positive or negative charge and it is this charge difference that helps drive chemical reactions to keep us all alive &ndash that&rsquos important. Anions are those elements or molecules that in their natural state have a negative (-) charge. Cations are those that in their natural state have a positive (+) charge. Negative charge, positive charge &ndash who cares? Keep reading.

Most soil particles have a negative charge. The amount of negative charge depends on soil texture, such as sand, silt and clay content, which is directly related to soil particle surface area. The cation exchange capacity (CEC) determined by a soil test is a direct indication of the amount of negative charges on your soils. A soil with low CEC has fewer negative charges than a soil with a higher number. High sand soils generally have a low CEC, clay or silt soils are higher and organic soils are highest &ndash all related to particle surface area.

Now the important part! Since soils are negatively charged and plant nutrients are positive and negative, some nutrients are attracted to soil while others are not &ndash the &ldquoopposites attract&rdquo principle. Those nutrients that exist as anions (-) are moved through soil, meaning growers need to be careful how they are applied regardless of soil type. These nutrients readily travel wherever water carries them, leading to nutrient runoff and leaching and economic loss and environmental concern.

Cations (+) are more readily bound to soil, resulting in these nutrients moving through the soil more slowly. However, since low CEC soils have fewer negative charges, cations will move more quickly through low CEC (sandy-based) soils than they will through high CEC (loamy and silt/clay-based) soils.

All this positive-negative, cation-anion, high CEC-low CEC stuff comes into play when applying nutrients and water. Table 1 gives the soil-borne elements necessary for plant growth, the form taken up by the plant and the element&rsquos mobility in the soil. Note that most mobile elements have a negative charge and the somewhat mobile and immobile elements have a positive charge. Over application of a (-) charged element followed by excessive water will quickly move that element through the system. Likewise, over application of most (+) charged elements on a low CEC soil can move that element through the system since there are not enough (-) charges on the soil particle surface to bind to the cation.

The odd anion is phosphorous. Even though it has a (-) charge, it is not mobile in soil because phosphorous forms are not very soluble. It can, however, still move &ndash not as the anion, but bound to soil particles as the particles move. Therefore, minimizing runoff is helpful in reducing phosphorus pollution.


Using Nutrients to Prevent Plant Problems

In some cases, plants suffer from certain illnesses &ndash fungal, viral, bacterial, and more &ndash due to a lack of certain nutrients in their environment.

As with people, the intake of certain nutrients can be a key to forestalling illness, like vitamin C&rsquos associations with reducing symptoms of the common cold.

How does this happen with plants? Let&rsquos take a look.

Fungal Plant Diseases

Downy mildew, fusarium, and others directly invade plant tissues that are weak from a lack of nutrients. A rise in fungal illness may signify the need for calcium, potassium, or phosphorus.

Viral Plant Diseases

Excess of certain nutrients (especially nitrogen and phosphorus) can increase susceptibility to viruses. This can sometimes be balanced with more potassium.

Bacterial Plant Diseases

After adding any amendment, wait a week or two and watch for improvement. If it&rsquos not obvious that your plants are getting healthier, then you may want to diagnose for other problems just to be safe.

Low calcium, nitrogen, and potassium can make your plants susceptible to bacterial illnesses, as described by the University of Florida Extension. On the other hand, too much nitrogen can help certain bacteria to thrive as well.

If you encounter any of the above categories of illness, try balancing the nutrients in your soil by following each respective suggestion.

After adding any amendment, wait a week or two and watch for improvement. If it&rsquos not obvious that your plants are getting healthier, then you may want to diagnose for other problems just to be safe.

If plants fail to recover, take it as a sign that they are too diseased and unhealthy to improve &ndash and remove them from your garden quickly if that is the case.

Pests

A lack of proper plant nutrition can have a big impact on the influence of pests.

It&rsquos a no-brainer that a nutrient-depleted, unhealthy field will be much more vulnerable to pests, in comparison to a perfectly nutrient-healthy field &ndash as studied and tested in this article from Ag-USA.net.

Some nutrient factors in the soil may contribute to the increased likelihood of undesired pest outbreaks. Excessive nitrogen is the most notable: studies cited by eXtension.org reveal that too much leads to increased pest populations of arthropods (i.e. aphids, mites, etc.), so take care not to go overboard!

Weak, undernourished plants also send out chemical signals to pests that they are languishing &ndash thus, depleted plants without adequate nutrition quickly hasten their own end.

On the other hand, healthy plants with plenty of nutrients do just the opposite, attracting more beneficial bugs than harmful varieties, even some that are ready to dine on nearby pests to prevent damage!

For that reason, providing a full range of nutrients for your plants is the best way to prevent pests as organically as possible.


Plasticity in nitrogen form uptake and preference in response to long-term nitrogen fertilization

Niche complementarity arising from divergence in resource use is an important mechanism underlying species coexistence. We hypothesized fertilization with different N forms would generate plastic divergence among species with regard to their N form uptake and preference.

Methods

In the eighth year of a long-term N fertilization experiment in an alpine meadow on the Tibetan plateau, we labeled 11 common plant species with ammonium- 15 N or nitate- 15 N in subplots without fertilization (control) or fertilized with 7.5 g N m −2 yr −1 in the form of ammonium, nitrate, or ammonium nitrate to trace N form uptake.

Results

Depending on species, fertilization with nitrate or ammonium nitrate had positive, negative or neutral effects on NO3-N uptake rate, although ammonium fertilization showed little impact. By contrast, fertilization with any N form had little impact on NH4-N uptake rate. Consequently, effects of nitrate fertilization and ammonium nitrate fertilization on relative N form preference diverged among the species and the functional groups (grasses, sedges, legumes and forbs).

Conclusions

Alpine plant species can diverge in N form uptake and preference in response to long-term N fertilization, and such divergence may contribute to species coexistence after long-term fertilization.


Discussion

In this study, our main motive was to investigate the PGP abilities of endophytic bacterial strains isolated from lodgepole pine trees growing in a nutrient-poor, disturbed ecosystem in the SBPSxc region in BC. When the six endophytic bacterial strains were analyzed for their potential to enhance tree growth via inoculation studies with their original host (lodgepole pine) and a foreign host (hybrid white spruce), it was observed that all strains were effective in significantly increasing the length (30–60%) and biomass (125–302%) of both tree hosts, 540 days after inoculation (Figs 1 and 2). Comparable growth promotion has been observed in previous greenhouse studies conducted for a similar time period, in which endophytic PGPB were inoculated into Pinaceae trees such as lodgepole pine, Douglas-fir, Scots pine, western red cedar and hybrid white spruce [11, 26, 49–52]. However, the growth promotion observed for our strains may be overestimated and should be interpreted with caution since the pine and spruce seedlings were grown under sterile conditions in a greenhouse set-up which is far from natural edaphic conditions.

Forming a close association with their host is a major strategy employed by PGPB to stimulate plant growth and health [53]. This was true for bacteria tested in this study since all strains had formed ten thousand to ten million colonies per gram tissue in the rhizosphere and internal root tissues of spruce and pine seedlings 540 days after inoculation (Fig 3). Internal stem colonization was also detected for all bacterial strains however, needle colonization was observed for a subset of strains, which indicates niche preferences exerted by either the bacterium or the tree host [54]. The rhizospheric and endophytic population sizes observed in our study align with those observed for other PGPB in coniferous as well as deciduous tree species [26, 32, 55–58]. However, plant colonization by our bacterial strains may vary significantly in natural field conditions when faced with significant abiotic and biotic stresses. Entry to and survival in internal plant tissues is facilitated by the secretion of protease and cellulase enzymes since these enzymes can disintegrate and metabolize plant cell wall polymers, proteins and other organic compounds in the apoplast [59, 60]. Since all strains possessed the ability to secrete at least one of these enzymes (Tables 1 and 2), it can be suggested that internal tissue colonization may have resulted due to the functioning of these enzymes. It is interesting to note that four strains that showed the presence cellulase and protease enzymes were the only ones that were able to colonize the needle tissues of both pine and spruce (Fig 3), potentially indicating the combined role of these enzymes in helping the bacteria to enter, move, survive and multiply in plants, particularly in the needle tissues.

The rhizospheric and endophytic bacterial population for each strain in pine and spruce had a strong correlation (R 2 > 0.87) with seedling length and biomass (Fig 4). This suggests that the number of colonies formed by each bacterial strain directly affected their efficacy to enhance the growth of pine and spruce seedlings. Similar observations have been reported in inoculation studies with interior spruce, lodgepole pine, poplar, corn, canola and tomato [32, 56, 61–63]. The best plant colonizers in our greenhouse growth trials–C. sordidicola HP-S1r, C. udeis LP-R2r and P. phytofirmans LP-R1r –were the best plant growth promoters as they promoted seedling length by up to 60% and seedling biomass by up to 302% (Fig 4). However, it is difficult to determine whether this plant-growth-promotion effect was due to the rhizospheric population or endophytic population, or due to a synergistic effect of both populations, therefore further research focusing on this subject is necessary.

Phytohormones like IAA and ethylene play a crucial role in the growth and development processes of a plant such as seed germination, root development and proliferation, stem and root elongation, reproduction, and fruit ripening [23]. Modulation of these hormones by PGPB living in close association with the host plant is a well-known phenomenon to enhance the growth and health of the host plant in exchange for energy for the microbes [64]. For instance, when the endogenous production of IAA by plants is insufficient to support their growth and development, reliance on exogenous IAA produced by associative PGPB can be a viable alternative [28, 65]. Such PGPB convert L-tryptophan, a metabolite commonly present in plant exudates, into IAA indicating the development of a mutually-beneficial relationship between the PGPB and the host plant. The ability to convert L-tryptophan to IAA was confirmed in all of our strains via in vitro broth assay (Table 2). Since all strains were also observed to significantly enhance the length and biomass of pine and spruce seedlings in the greenhouse trials (Figs 1 and 2), our results are consistent with the theory that IAA production leads to greater elongation, proliferation and development of the plant tissues [23, 64, 66]. In particular, the highest IAA producing strains–C. sordidicola HS-S1r, P. phytofirmans LP-R1r and C. udeis LP-R2r –were also the best performing strains in the greenhouse which further support this theory. However, further evidence is required to confirm the IAA-producing ability of these strains in planta by raising IAA-negative mutants and then comparing the release of IAA in plant roots after inoculation with IAA-positive versus IAA-negative derivatives. In addition, in planta expression of genes related to IAA production such as iaaH and iaaM genes could be performed using qRT-PCR to detect the relevant gene copy numbers. The significant plant growth promotion observed specifically under nutrient-stress conditions in the greenhouse can also be linked to the ability of all of our strains to modulate plant-ethylene levels by releasing ACC deaminase (Table 2). Plants produce excess amounts of ethylene when subjected to stress conditions which can inhibit their growth and development [67]. However, certain associative PGPB produce ACC deaminase to cleave ACC–the precursor of plant-ethylene–and convert it to α-ketobutyrate and ammonia, thereby reducing excess plant-ethylene levels [23, 44]. ACC deaminase producing bacteria utilize the ammonia produced from this reaction for their own metabolism. We used a binary approach–in vitro enzyme assay and in vivo gnotobiotic assay–to evaluate the ACC deaminase activity of our bacterial strains. The amount of ACC converted to α-ketobutyrate by our bacterial strains (33–111 nmol/mg/hr) in the in vitro enzyme assay was analogous to the typical range observed in previous studies with endophytic bacteria [68–71]. All bacterial strains also showed positive ACC deaminase activity when inoculated into ethylene-sensitive plants–canola and tomato–in the in vivo gnotobiotic assay (Fig 5). It should be noted that a uniform and strong correlation (R 2 > 0.75) between the in vitro and in vivo analyses of ACC deaminase activity was observed for our strains (Fig 6). The primary root length of inoculated canola and tomato seedlings was 2-fold or higher than non-inoculated control seedlings, which is consistent with the findings of Anandham et al. [72] and Onofre-Lemus et al. [73]. It has been postulated that alleviation of excess plant-ethylene levels after seed germination by ACC deaminase producing bacteria is a priming effect to significantly enhance the root length of inoculated seedlings after germination. Although excess ethylene levels are required to break seed dormancy, high levels after germination could lead to stunted growth. However, ACC deaminase producing bacteria could negate this effect [23, 44]. Although our strains showed significant results for in vivo root elongation–suggesting the synthesis of ACC deaminase enzyme–however, to confirm this, in planta acdS gene expression of these bacterial strains needs to be evaluated. Additionally, specific proteins secreted by each bacterial strain to cleave ACC need to be identified so as to confirm the secretion of these proteins in planta following inoculation.

Lytic enzymes, including cellulase, protease, chitinase and β-1,3-glucanase, represent a major category of secondary metabolites released by PGPB to lyse cell walls of plants and microorganisms as well as cuticles and eggshells of pests like nematodes which may lead to suppression of phytopathogens and/or decomposition of litter and nutrient turnover [74–76]. All of our strains were tested positive for the presence of at least one of these four enzymes in both qualitative and quantitative assays, with comparable results observed for each strain in both assay types. The enzyme units of cellulase, protease, chitinase and β-1,3-glucanase produced by our strains fall within the usual range detected for rhizospheric and endophytic PGPB [37, 77–80]. However, the enzyme activity detected under in vitro conditions should be interpreted with caution because the ‘actual’ production of these enzymes may vary under in vivo conditions where a variety of external factors can affect the syntheses of these enzymes. Methods such as in situ gene expression and proteomics needs to be applied to confirm the activity of these enzymes in real plant conditions. In addition, the antagonistic potential of these strains against plant pathogens must be evaluated through co-inoculation of these strains with a pathogen in plant to confirm if the production of these enzymes ‘actually’ leads to the suppression of the pathogen. Each lytic enzyme has a specific function to degrade particular compounds in the cell walls and cuticles. For example, cellulase enzyme degrades the glycosidic linkages in cellulose chains that form intra- and intermolecular hydrogen bonds, protease enzyme breaks the peptide bonds present in the protein matrix, chitinase enzyme disintegrates the rigid chitin polymer, and β-1,3-glucanase enzyme degrades the β-1,3-linked backbone of glucan, a cell wall polysaccharide [78]. Notably, the activity of all four enzymes was detected only in C. sordidicola HS-S1r, P. phytofirmans LP-R1r and C. udeis LP-R2r, which indicates that these strains may be effective at controlling phytopathogens and/or decomposition by possibly exerting a synergistic mechanism. Another common biocontrol mechanism includes the production of toxic compounds like ammonia gas by PGPB [81]. All of our strains possessed the ability to produce ammonia gas as observed by the formation of yellow/brown colour when Nessler’s reagent–a common ammonia-detecting compound–was added to the broth. In comparison to other strains, the development of darker brown colour for P. phytofirmans LP-R1r indicated the highest ammonia production by this strain. All bacterial strains except P. frederiksbergensis HP-N1r were observed to secrete catalase (Table 1), which is an enzyme known to neutralize the overproduction of reactive oxygen species (ROS) under stress conditions. However, in vitro production of cellulase, protease, chitinase, β-1,3-glucanase and catalase enzymes cannot be related to their production in a plant system and further studies need to be conducted to observe the production of genes related to these enzymes via qRT-PCR. In addition, proteins relevant to these enzymes secreted by our strains need to identified, so that production of these enzymes could be confirmed in the plant-soil system after bacterial inoculation.

Siderophore production in combination with the ability to synthesize lytic enzymes and toxic gases is believed to be extremely lethal against phytopathogens, particularly fungi [82–84]. In addition to efficiently chelating the Fe 3+ molecules present in soil and depriving the pathogens from taking up iron, siderophores produced by PGPB are also responsible for triggering ISR in plants [85–88]. All but one strain showed positive siderophore production in the range observed for PGPB isolated from natural and cultivated ecosystems [24, 26, 28, 82, 88, 89]. Along with assisting the plant in mediating biotic stress, PGPB-produced siderophores can also help the plant in acquiring ferric iron, a scarce micronutrient that mostly exists in plant-unavailable form in soils [90]. PGPB can also facilitate plant acquisition of macronutrients like phosphorus via two major mechanisms–solubilization of inorganic phosphorus and mineralization of organic phosphorus [91]. Since the scarcity of plant-available phosphorus in soils is often an issue in both agriculture and forest ecosystems, the presence of PGPB that can effectively convert the unavailable forms of phosphorus in the soil to available forms is vital for sustainable productivity [23]. In vitro plate and broth assays revealed that all but one bacterial strain possessed both inorganic phosphate solubilizing and organic phytate hydrolyzing abilities (Tables 1 and 2). For inorganic phosphate solubilization, the SI observed in the plate assay (1.2–2.6) and the amount of soluble phosphates released in the broth assay (66–112 μg per mL broth) by our bacterial strains align with previous studies in which PGPB isolated from crop and tree hosts with strong phosphate solubilization abilities have been reported [24, 26, 28, 37, 92–94]. The mechanisms of inorganic phosphate solubilization mainly include the production of organic acids, protons, siderophores and exopolysaccharides [91]. Although several organic acids could be released by PGPB, gluconic acid represents one of the most common and effective forms of organic acid responsible for releasing phosphorous [95]. Since the major source of phosphorus in forest ecosystems is organic litter, the ability to produce phytase to mineralize certain forms of organically-bound phosphorus is crucial for the growth of trees. As plants are not known to directly take up phytate from the soil or mineralize it, phytase-secreting microbes have an important role to play in hydrolyzing phytate and making it available for plant uptake [91, 96]. The five strains that hydrolyzed Na-phytate in both qualitative plate assay and quantitative broth assay (Tables 1 and 2) performed similarly well in comparison to previous studies [38, 39, 96, 97]. All in all, the five strains that possess the ability to make both inorganic and organic phosphorous available for plants could be referred to as comprehensive phosphate solubilizing bacterial strains. In particular, C. udeis LP-R2r which had significantly higher inorganic and organic phosphate solubilizing ability than all other strains should be further evaluated to quantify the amount of phosphorus made available by this strain in planta. Furthermore, additional data needs to be collected to confirm the increased phosphorus uptake by plants when inoculated with these strains using stable isotope method, gene expression analysis and proteomics.

Since both lodgepole pine and hybrid white spruce are conifer species that have been reported to associate widely with mycorrhizal fungi in natural temperate and boreal forests, it is inevitable to sideline their role in tree growth promotion. In a previous study, lodgepole pine and other Pinaceae tree species including interior Douglas-fir (Pseudotsuga menziesii) and ponderosa pine (Pinus ponderosa) inoculated with isolates of Laccaria laccata, Rhizopogon vinicolor and Suillus luteus showed a significant increase in height (up to 23%) and root collar diameter (up to 45%), 2 years after inoculation [98]. Intriguingly, authors confirmed that the observed growth responses of conifer seedlings were partially influenced by IAA and ethylene produced by these ectomycorrhizal fungal symbionts [98]. In another study, isolates of ectomycorrhizal fungus Hebelomaarenosa arenosa were reported to enhance the shoot height and root dry weight of red pine (Pinus resinosa) when grown without fertilizer applications in a greenhouse [99]. These fungal isolates also increased the survival rate of red pine seedlings after outplanting into field conditions. Similarly, above-ground growth of radiata pine (Pinus radiata) seedlings was significantly enhanced by inoculation with Rhizopogon roseolus and Scleroderma citrinum isolates, 2 years after outplanting onto low soil moisture sites in Spain [100]. It is interesting to note that ectomycorrhizal fungi and PGPB have been observed to work synergistically to promote the growth of lodgepole pine and hybrid white spruces seedlings in greenhouse studies indicating that their combined capability could be sufficient to support the growth of Pinaceae trees on disturbed sites [101, 102]. Therefore, our PGPB strains should be evaluated with mycorrhizal fungi in future field-based studies to determine their ‘real’ benefits and evaluate if they work coherently with mycorrhizal fungi to sustain the growth of Pinaceae trees in the SBPSxc region.

In conclusion, the six bacterial strains evaluated in this study can colonize the rhizosphere and internal tissues of multiple Pinaceae trees–lodgepole pine and hybrid white spruce–and enhance their growth by potentially exerting diverse PGP mechanisms. Of the 11 mechanisms tested in this study, all bacterial strains were tested positive for at least 5 different mechanisms involving key PGP traits. Notably, three bacterial strains–C. sordidicola HS-S1r, P. phytofirmans LP-R1r and C. udeis LP-R2r –possess the highest potential to promote plant growth using all mechanisms tested in this study. This impressive suite of in vitro PGP capabilities could be related to the significant enhancement of pine and spruce length and biomass observed for these strains in the greenhouse growth trial. Bacterial strains belonging to Caballeronia and Paraburkholderia genera possess multiple PGP abilities as reported in previous studies using lab-based enzyme assays, genomic analyses, and in planta assays [9, 33, 103–105]. Intriguingly, these genera were previously assigned to the plant-beneficial group of the Burkholderia genus [106, 107], which is rich in potent plant-probiotics [21, 108]. Although we have demonstrated the growth-promoting potential of our strains in the greenhouse experiment, the in vitro PGP experiments do not provide enough evidence for the mechanisms behind the observed growth promotion. The in vitro conditions in which the PGP abilities of our bacterial strains were observed represent highly sterile and simulated conditions, therefore functioning of these PGP mechanisms must be validated under realistic conditions in planta. Potential approaches may include raising negative mutants lacking the relevant PGP trait and comparing it with the wild-type strain via plant inoculation studies, conducting whole genome sequencing of these bacterial strains to identify their PGP-related genes and then evaluating the expression level of PGP-related genes in planta following inoculation, and co-inoculation of plant with a pathogen and our PGP strains to observe their ability to secrete various lytic enzymes and suppress pathogen in natural conditions. Considering the results of this study and a previous report where these bacterial strains showed considerable nitrogen-fixing ability [5], we can conclude that such strains with multifarious PGP abilities may be playing a significant role in sustaining the growth of Pinaceae trees under nutrient-limited, disturbed edaphic conditions of the SBPSxc region. Henceforth, such bacteria with the inherent ability to enhance the growth of multiple tree hosts should be further evaluated in the field to determine their actual benefits under natural conditions.


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