Phosphorus (P) is one of the 16 elements essential for plant growth [1
]. Phosphate availability greatly determines growth and fitness of the plants and thus crops quality and yields [2
]. In condition of P deficiency, plant root development is inhibited and this leads to a delay in plant growth [3
]. Phosphorus concentration in natural soils varies from 50 to 3000 mg kg−1
of soil, yet only 0.1% of total phosphorus is really accessible to plants [4
], Indeed, most phosphate is immobilized in the soil [6
] either via its adsorption on soil particles, precipitation with various minerals (Al, Fe and Ca) or interconversion into organic forms by soil-born micro-organisms [7
]. To overcome this problem, nearly two millions tons of soluble chemical phosphate fertilizers are spread each year on agricultural fields worldwide [8
]. However, still a significant fraction of these fertilizers is converted into insoluble forms [9
The increasing awareness of environmental issues linked to agrochemical inputs stimulates the development of a sustainable agriculture and the replacement or complementation of the chemical fertilizers by other more ecological and environmentally friendly processes [10
]. The direct use of natural rock phosphate in traditional agriculture in Morocco is one of such processes [11
]. Morocco contains three quarters of the world’s rock phosphate (RP) reserves [12
]. RP is an hydroxyapatite (Ca10
) not directly usable by plants [13
] except in some acidic soils or in soils rich in specific micro-organisms able to convert insoluble phosphate into a form easily assimilable by plants [14
]. Several reports demonstrated that these P-solubilizing microorganisms (PSM) could increase growth and crops yields of several agricultural plants including wheat [15
], chickpea [16
], rice and tomato [17
] and can potentially be used as P-biofertilizers.
Sugar beet(Beta vulgaris
L.) is the main industrial crop grown in the vast agricultural lands of the Beni-Mellal region of Morocco and constitutes 21.2% of the national production of sugar beet [18
]. Since increasing growth and yield of this economically important crop is a constant concern, we investigated the presence of PSM endemic to these specific soils.
Among these PSM, Actinobacteria, including Actinoplanes, Streptomyces and Micromonospora
], are of special interest since besides their PSM abilities, they also produce bioactive secondary metabolites able to limit growth of various phytopathogens agents [9
] or molecules stimulating growth or eliciting natural plant defenses [23
]. Exploring the richness of endemic PSM Actinobacteria in the soils specifically used for sugar beet cultivation is of interest to develop adequate new bio-fertilizer agents to stimulate sugar beet growth in the Beni-Mellal region. We thus screened for and isolated Actinomycetes endemic to these sugar beet rhizospheric soils that were able to grow and release soluble phosphate from insoluble phosphate sources in laboratory conditions. Putative solubilization mechanisms used by these bacteria were discussed and taxonomic characterization of the most efficient solubilizing isolates was achieved.
3.1. Soil Analysis
Physico-chemical proprieties of soil samples are listed in Table 1
. The tested sugar beet agricultural soils contained, on average, 5% of Organic Matter (OM) and 86% of Mineral Matter (MM) including 2% nitrogen, 3.5% P2
, 0.35% K2
O, MgO (1.4%) and MnO (0.08%). The pH of the soils was close to neutrality or slightly basic (pH 7.4 on average). The highest electrical conductivity indicating soil salinity was recorded in soil sample 2 with an average of 0.47 (µS/cm).
3.2. Distribution of Total Flora and of Actinomycetes in the Sugar Beet Fields under Study
The distribution of total flora (TF) and Actinomycetes of sugar beet soils collected from the three sites is shown in Table 2
. TF was at a similar level in the three sites (on average 23 × 107
cfu/g of soil) (Table 2
). The Actinomycete isolates were significantly more abundant in sites 1 (7.2%) and 3 (6.73%) than in site 2 (4.90%) (Table 2
Among the 164 Actinomycete isolates retained, 57 had the ability to grow on SMM + RP as the sole phosphate source. Among these 57 isolates, only 27 isolates (47.36%) had also the ability to grow on SMM + TCP as unique P source. Ten of the twenty-seven isolates, showing the highest biomass yield on SMM + RP and with different morphological characteristics, were selected for further studies. Seven of these ten Actinomycete isolates were from site 1 (AI, AYD, AV, AZ, BYC, BX, and BP), one (CYM) from site 2 and two from site 3 (DE1 and DE2).
3.3. Growth Kinetic of the Selected Actinomycete Strains in SMM + RP and SMM + TCP
clearly shows that the growth kinetics of most strains was similar in both TCP and RP. This indicated that the strains were able to assimilate similar amounts of phosphorus from these phosphate insoluble sources, with a comparable efficiency, and use it for their own growth. The only exception was the strain BP that showed a better growth on SMM + RP than on SMM + TCP. One notes that, in SMM+ TCP mainly, most strains yielded a lower biomass at day 5 than at day 4, suggesting cell lysis. However, the biomass yield was not the same for all strains and the strains could be grouped into two classes, class I with biomass yield above 70 µg/mL (DE2, BYC, AYD, AZ, AI and BP) and class II with biomass yield comprised between 50 and 70 µg/mL (AV, DE1, CYM and BX) (Figure 3
). One notes that strains with the lowest biomass yields are also those yielding the lowest amount of soluble phosphate (Figure 3
3.4. Estimation of the Amount Soluble Phosphate Released from TCP and RP by the Selected Actinomycete Strains
The concentration of free phosphate spontaneously released from TCP and RP in the control non-inoculated flask was 2.5 and 5 µg/mL, respectively. The concentration of soluble phosphate in the supernatant of most strains (except perhaps CYM and BX), exceeded this value (Figure 3
). This indicated that most strains were able to release phosphate from these two different insoluble phosphate sources in excess of their phosphate need to support their growth. However, the amount of soluble phosphate released greatly varied with the nature of the phosphate source and from strain to strain.
Five strains released a high (>60 µg/mL) and higher amount of phosphate from TCP than from RP (Figure 3
), indicating that TCP was more efficiently solubilized than RP. The presence of large amounts of phosphate in the supernatant of these strains in the presence of TCP simply indicated that the rate of Pi released from TCP exceeded its rate of consumption for bacterial growth.
Strain DE2 (class I) released maximal soluble phosphate concentration (180 µg/mL) from TCP, whereas strains BYC (class I) and BP (class I) released maximal amounts of soluble phosphate from RP (148.05 µg/mL and 59.44 µg/mL, respectively).
The strain BYC (class I) is of special interest since it was able to release a similar amount of Pi from RP (150 µg/mL) and TCP (170 µg/mL) and its biomass yield was similar with both phosphate sources (only12% higher in SMM + RP than in SMM + TCP). The strains BP (class I) and DE1 (class II) released over 2 fold more soluble phosphate from RP (60 µg/mL and 15 µg/mL, respectively) than from TCP (25 µg/mL and 7 µg/mL, respectively). The biomass yield of the strain BP was 20% higher in SMM + RP than in SMM + TCP whereas that of the strain DE1 was similar on both P sources. Interestingly, the strains BX (class II) and CYM (class II) consumed the totality of the phosphate released. This suggested that the solubilizing ability of these strains was less efficient than that of the others.
3.5. Cas-Agar Test and Evolution of the pH of the Growth Medium
The CAS-agar test indicated that the six more efficient TCP solubilizing Actinomycete strains (DE2, AYD, BYC, AZ, AI, BP) were producing siderophores (Figure 4
) as previously reported for other PSM bacteria [20
]. However, among these strains only BYC was able to efficiently release phosphate from RP. The strains BX, AV, DE1 and CYM apparently produced little siderophores (Figure 4
) and were among the strains releasing very little phosphate from TCP or RP (Figure 3
). This suggested that the production of siderophores contributes to the P solubilization process.
The pH of the growth medium of all strains in TCP as in RP felt between 6 and 6.5 at day 1 and raised afterwards in most cases. This indicated that the earliest solubilization process might involve the excretion of organic acids. The pH of the medium remained below 6 for the strains DE2, AZ and BX in TCP for the following days but reached 7 or above in RP. DE2 and AZ release a fair amount of P from TCP (180 µg/mL and 100 µg/mL, respectively) but a rather weak amount from RP (20 µg/mL) whereas BX did not release any P from any of these phosphate sources. DE2 and AZ possibly excreted more siderophores than BX (Figure 4
In RP, the pH of the medium remained above 7 for most strains except for BYC (pH between 6.5 and 6.8 at days 2, 3 and 4) and CYM (pH 6 at days 1 and 2). BYC released a fair amount of phosphate from RP as well as from TCP (150 µg/mL and 170 µg/mL, respectively) whereas CYM hardly released any P from these two phosphate sources. BYC, but not CYM, was shown to excrete siderophores (Figure 4
The pH of the medium of the 5 remaining strains (AYD, BP, AV, DE1 and AI) was rather similar in TCP and RP and above 7. AYD and AI, produced siderophores and released a fair amount of phosphate from TCP (140 µg/mL and 60 µg/mL, respectively) but a rather weak amount from RP (20 µg/mL). BP that is producing siderophores released more phosphate from RP (60 µg/mL) than from TCP (24 µg/mL). The latter two strains AV and DE1 were weak siderophore producers and yielded weak amount of phosphate from TCP (18 µg/mL and 7 µg/mL, respectively) as well as from RP (10 µg/mL and 14 µg/mL, respectively).
3.6. Taxonomical Characterization of the Selected Isolates
In order to determine whether the 10 isolates were similar or different strains, their ability to assimilate 10 different carbon sources was tested. Most strains were able to use mannitol, lactose, glucose, fructose, maltose, galactose, sucrose, sorbitol and glycerol as sole carbon sources, except AV that did not use mannitol, lactose, maltose, fructose and sorbitol; DE2, BYC, BP, AV and BX that did not use fructose; AYD that did not use maltose (Table 3
) and DE2, BP and BX that did not use nor maltose nor fructose (Table 3
). This preliminary analysis suggested that these strains were likely to be different.
These 10 isolates were also evaluated for their ability to withstand salt stress by growing at NaCl concentrations of 5, 7 and 10 g/L. All strains showed the best growth at 5 and 7 g/L NaCl (Table 3
) except DE2, AYD and DE1, while CYM and BX showed better growth at 10 g/L NaCl (Table 3
). Therefore, these strains could potentially be halotolerant. The analysis of cellular constituents of all isolates revealed the presence of L- diaminopimelic acid (DAP) isomer (Table 3
), confirming that they belong to the Streptomyces
The sequences of 16S rRNA gene of the 10 strains were analyzed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST
accessed: 29 March 2021) and the LEBIBI [39
] and GenBank databases. They were all found to belong to the Streptomyces
genus bearing an identity of at least 99%. Nucleotide sequences of partial 16S rRNA of the identified isolates were deposited into Gen-Bank Database (http://www.ncbi.nlm.nih.gov/GenBank/
accessed: 29 March 2021), under the accession numbers listed in Table 4
16S rDNA sequences of Streptomyces
species retrieved from Genbank as well as that of our strains were used for the construction of a phylogenic tree (Figure 5
). Six strains (BP, BX, DE1, AV, AYD and AZ) were closely related to Streptomyces bellus
, AI was related to S. tunisiensis
, BYC to S. enissocaesilis
, DE2 to S. saprophyticus
and CYM to S. cyaneofuscatus
Most publications describing isolation of phosphate-solubilizing bacteria (PSB) used growth on TCP, rather than on RP, but analysis of scientific literature concerning biological P solubilization suggested that this substrate might not be the most appropriate to screen for PSB able to enhance plant growth [45
]. In consequence, we instead used growth on RP as first screen to isolate PSM and our strategy and outcomes are summarized in Figure 6
Our study revealed that 28% of the Actiomycetes isolated from sugar beet soils had the ability to grow on RP as sole phosphate source and among them only 47.36% had also the ability to grow on TCP as sole phosphate source. This difference is difficult to explain but suggested that the TCP and RP solubilizing processes may involve different mechanisms or that the greatest diversity of mineral elements present in RP compared to TCP, is necessary for the growth of some strains.
Five strains (DE2, BYC, AYD, AZ and AI) among the ten strains studied were able to release significant amount of phosphorus from TCP (>60 µg/mL). Their solubilization activity was comparable to that of Azospirillum
sp., Pantoea agglomerans
and Pseudomonas fluorescens,
from wheat rhizosphere in Jensen medium [47
]; however, it was much higher than that reported for a Streptomyces
sp. (AH6 strain) isolated from the rhizospheric soil of Calluna vulgaris
However, among these five efficient solubilizing strains, only one strain, BYC related to Streptomyces enissocaesilis
), was able to release similar amount of phosphorus from RP and TCP and another strain, BP, was able to release even more phosphate from RP than from TCP. These two strains are thus of special interest and the mass spec analysis of the molecules present in their supernatant is expected to lead to the purification and structural characterization of potentially novel siderophores and/or organic acids contributing to their efficient RP solubilization process.
In RP the pH of the medium of all strains, except that of BYC, was between 7 and 8, suggesting that the solubilization process does not involve the excretion of organic acids but rather that of siderophores [41
]. The very efficient solubilization process of BYC might involve both the acidification of the growth medium and the excretion of siderophores.
In TCP, the growth medium of DE2 and AZ turned out to be acidic but that of the other strains was close to or above 7. This suggested that the TCP solubilization process of DE2 and AZ might involve the excretion of organic acids. To our knowledge, that is the first report of Actinomycete strains solubilizing insoluble P via the production of organic acids. In this process, negatively charged organic acids are thought to chelate Ca2+
counter ions of phosphate [43
]. A similar process was reported in other fungi such Penicillium aurantiogriseum
] and Penicillium radicum
In conclusion, we anticipate that our most efficient RP solubilizing strain, BYC, produced in large scale may constitute a novel kind of fertilizers useful to solubilize the phosphate trapped in the soil to feed and stimulate sugar beet growth. Such strategy would contribute to the development of a bio-based economy supporting a sustainable and environmentally friendly agriculture.