Penicillium strains, compositions and methods of promoting plant growth

Abstract

The present invention generally relates to an isolated Penicillium strain, named Penicillium olsonii TLL1, with a preservation number of NRRL 68252, and to the use thereof in promoting plant growth, and to the use of the Penicillium strain, named Penicillium olsonii TLL1, named Penicillium olsonii TLL1, named Penicillium olsonii TLL1, named Penicillium olsonii TLL1. Specifically, the present invention provides compositions suitable for promoting plant growth comprising the isolated fungal strains that can mitigate Al toxicity 5 and increase phosphate effectiveness, as well as methods of their promotion of plant growth.

Description

[0001] Citation of biological material deposit

[0002] This application contains a citation to a biological material deposited under the Budapest Treaty, having the accession number NRRL 68252, deposited on January 18, 2023, at the Agriculture Research Service Culture Collection (NRRL), which is incorporated herein by reference. TECHNICAL FIELD

[0003] The present invention generally relates to an isolated Penicillium strain, designated Penicillium olsonii TLL1, having the accession number NRRL 68252, and its use in promoting plant growth. Specifically, the present invention provides a composition suitable for promoting plant growth comprising the isolated fungal strain that can alleviate Al stress and increase phosphorus availability, and a method of promoting plant growth thereof. BACKGROUND

[0004] The availability of macro- and micronutrients plays a crucial role in the growth and development of plants. Among the macro nutrients, phosphorus is an indispensable element involved in plant metabolic processes and signal transduction pathways. Despite being the 11th most abundant element in the Earth's lithosphere, 29% of the world's arable land is deficient in phosphorus. Low mobility in soil, precipitation as inorganic metal oxides, and immobilization in organic compounds make phosphorus inaccessible to plants. Phosphorus limitation is a major nutrient limiting factor as it affects plant growth and development and impairs optimal yield. The agricultural sector generally addresses phosphorus deficiency by the application of phosphorus fertilizers, but due to the rapid immobilization of phosphorus into insoluble forms, the efficiency of phosphorus utilization by plants is only 5-25%. Moreover, over-application leads to soil fertility decline and associated environmental hazards. Therefore, there is an urgent need to develop environmentally friendly strategies to increase the uptake of phosphorus by plants.

[0005] Phosphate-solubilizing microorganisms (PSM) help in the assimilation of phosphate from both organic and inorganic forms of phosphate in the soil, making it available for plant uptake. PSMs attribute the acquisition of soluble phosphate to a variety of mechanisms, such as chelation and ion exchange processes, production of organic acids, secretion of microbially derived enzymes such as acid phosphatases, phytases, and production of plant hormones (Bergkemper et al., 2016). In this regard, phosphate-solubilizing fungi (PSF), such as those from the genera Penicillium (Li et al., 2016), Aspergillus (Yin et al., 2017), and Trichoderma (Lei and Zhang, 2015), have been successfully used as microbial inoculants for various crops to improve phosphorus uptake, thereby increasing biomass and yield. For example, Penicillium oxalicum and Aspergillus niger have been shown to increase phosphate bioavailability and promote the growth of maize in calcareous soils (Yin et al., 2015), and Penicillium guanacastense has been reported as a potent biofertilizer that promotes the growth of Pinus massoniana under phosphorus-limiting conditions (Qiao et al., 2019). Clearly, in the current agricultural scenario, the use of efficient PSF is a promising eco-friendly strategy for improving phosphorus uptake by plants and promoting plant growth.

[0006] In addition to phosphorus deficiency, acid soils also severely affect the productivity of plants. While previously in early 1995 it was estimated that acid soils accounted for 50% of the world’s potentially arable soils, due to increasing air pollution and rising industrialization, the total area of acid soils worldwide is likely to have increased significantly. Aluminum (Al) toxicity in acid soils is one of the most important reasons for the significant decline in global crop productivity, and anthropogenic soil acidification caused by the widespread use of acid-producing fertilizers has accelerated the release of plant-toxic Al 3+ ions from the soil as they are readily soluble at pH < 5.0.

[0007] High concentrations of Al 3+ ions in the soil affect plant growth and productivity in multiple ways, including inhibition of root growth, reduced nutrient and water uptake. Impaired primary root growth and development is a major symptom of plant Al toxicity, which is mainly due to the inhibition of cell division and cell elongation in the apical meristem zone. Specifically, it has been shown that the accumulation of nitric oxide (NO), iron (Fe), and callose in the root tip contributes to the inhibition of primary root growth upon Al toxicity.

[0008] Many Al-tolerant yeasts and fungi are able to grow at concentrations as high as 100 mM. For example, Penicillium janthinellum reduces aluminum toxicity by synthesizing citric acid (Zhang et al., 2002). In the presence of P. janthinellum, Al-stressed tomato (Solanum lycopersicum) extract and translocate more Al to the shoots and roots while maintaining their growth compared to control plants with significant growth retardation (Khan et al., 2015). Similarly, employing Al-tolerant fungi can thus provide an effective strategy to alleviate aluminum stress and improve plant development.

[0009] Accordingly, there is a need to provide improved compositions and methods for promoting and enhancing plant growth to overcome or at least ameliorate one or more of the above disadvantages. SUMMARY

[0010] The present disclosure relates to a newly isolated Penicillium strain, designated Penicillium olsonii TLL1 (“POT1”), which is capable of promoting plant growth.

[0011] According to a first aspect, there is provided an isolated fungal strain of the species Penicillium olsonii, having the accession number NRRL 68252, deposited with the Agricultural Research Service Culture Collection (NRRL) on January 18, 2023.

[0012] According to a second aspect, there is provided a plant growth promoting composition comprising the isolated fungal strain of the first aspect, mycelium, hyphae, spores and / or cell-free culture filtrate thereof.

[0013] According to a third aspect, there is provided the use of the growth promoting composition according to the second aspect as a fertilizer.

[0014] According to a fourth aspect, there is provided a method of promoting plant growth, the method comprising applying an effective amount of the growth promoting composition according to the second aspect to a medium for plant growth, wherein the application of the composition promotes plant growth compared to a plant grown in an untreated medium.

[0015] According to a fifth aspect, there is provided a plant seed coated with the isolated Penicillium olsonii strain of the first aspect.

[0016] Advantageously, the novel strain of the present disclosure is capable of solubilizing phosphate in soil and increasing the availability of phosphorus for plant uptake. More advantageously, the strain of the present disclosure is also capable of alleviating aluminum stress and promoting plant growth by inhibiting negative components of root development, such as the accumulation of iron, callose and / or nitric oxide in root cells. These and other advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description. BRIEF DESCRIPTION OF DRAWINGS

[0017] The accompanying drawings illustrate implementations disclosed herein and are used for purposes of explanation and should not constitute an exclusive limit of the invention.

[0018] Figure 1 Phosphorus-solubilizing fungi identification and growth promotion of Arabidopsis POT1 under P-sufficient and P-limited conditions were depicted. (A) Phylogenetic tree generated from maximum likelihood (ML) analysis of ITS sequence data of phosphorus-solubilizing fungi POT1. The nodes of the tree show the Boot strap values of 1000 replicates. (B) Colony morphology of phosphorus-solubilizing fungi POT1. POT1 was cultured on (left) PDA and (right) MEA for seven days. The scale bar represents 1 cm. (C) Arabidopsis plants grown under full P conditions with and without POT1. Four-day-old plants were transferred to full P medium pre-incubated with and without POT1 for 10 days under in vitro conditions. The scale bar represents 1 cm. Bar graphs show primary root length (D), shoot fresh weight (E), root fresh weight (F) of Arabidopsis Col-0 plants co-cultured with POT1 for 10 days under full P conditions. Bar graphs show combined data from three independent experiments (n=3; unpaired two-tailed t-test, ***P<0.001; **P<0.01; *P<0.05). (G) Shoot P content of Arabidopsis plants co-cultured with and without POT1 after four weeks. Concentration of total phosphorus content in Arabidopsis shoots significantly increased after growth under full P in the presence of POT1 under in vitro conditions (n=3; unpaired two-tailed t-test, **P<0.01). (H) Arabidopsis plants grown under low P conditions with and without POT1. Four-day-old plants were transferred to full P medium pre-incubated with and without POT1 for 10 days under in vitro conditions. The scale bar represents 1 cm. Bar graphs show primary root length (I), shoot fresh weight (J), root fresh weight (K) of Arabidopsis WT plants co-cultured with POT1 for 10 days under low P conditions. Bar graphs show combined data from three independent experiments (n=3; unpaired two-tailed t-test, ***P<0.001; **P<0.01; *P<0.05). (L) Shoot P content of Arabidopsis plants co-cultured with and without POT1 after four weeks. Concentration of total phosphorus content in Arabidopsis shoots significantly increased after growth under low P in the presence of POT1 under in vitro conditions (n=3; unpaired two-tailed t-test, **P<0.01).

[0019] Figure 2Accumulation of callose and NO in POT1 -treated roots was depicted. (A) Callose accumulation in Arabidopsis roots under full P and low P conditions with and without inoculation of POT1. Four-day-old plants were transferred to full P medium pre-incubated with and without POT1 for 10 days under in vitro conditions. Callose accumulation in 1.5 cm root tips was analyzed using aniline blue staining. White arrows point to callose accumulation in the stem cell niche under phosphate-limiting conditions. Three independent experiments were performed and one representative experiment is shown. Scale bar 100 pm. (B) Nitric oxide (NO) accumulation in Arabidopsis roots under full P and low P conditions with and without inoculation of POT1. Four-day-old plants were transferred to full P medium pre-incubated with and without POT1 for 10 days under in vitro conditions. NO accumulation in 1.5 cm root tips was analyzed using DAF-FM DA staining. Inoculation of POT1 reduced NO accumulation in roots. Scale bar 100 pm.

[0020] Figure 3 POT1 solubilization activity of insoluble phosphate under different sparingly soluble P sources was shown. (A) POT1 produced solubilized circular phosphorus zones when cultured on Pikovskaya agar and NBRIP medium amended with CaP for seven days. (B) Bar graph showing the phosphate solubility index of POT1 when cultured on Pikovskaya agar and NBRIP medium amended with CaP for seven days. (C) Phosphate solubilization capacity of POT1 in different inorganic phosphorus compounds such as tricalcium phosphate (CaP), aluminum phosphate (AlP), ferric phosphate (FeP), and hydroxyapatite (HA) after fourteen days. Scale bar represents 1 cm. (D) Soluble P concentration produced by POT1 in different inorganic insoluble phosphate such as CaP, AlP, FeP, and HA. POT1 spore suspension was inoculated in 100 ml of Pikovskaya medium amended with CaP, AlP, FeP, and HA for two weeks and soluble phosphate concentration was measured using phosphomolybdenum spectrophotometry. Each treatment included three biological replicates each containing three technical replicates (n = 3; unpaired two-tailed t-test, ***P < 0.001; **P < 0.01; *P < 0.05). Error bars show standard deviation.

[0021] Figure 4POT1 promotes growth of Arabidopsis under insoluble phosphate and phosphate limiting conditions. (A) Representative images of Arabidopsis WT plants grown with and without POT1 under full P, low P, CaP, AlP, FeP and HA as sole P sources. Ten-day-old plants were transferred to POT1 pre-inoculated and non-inoculated nutrient-poor vermiculite soil and harvested after two weeks. Scale bar represents 1 cm. Bar graphs represent shoot fresh weight (B), leaf area index (C), leaf number (D), shoot phosphate content (E) of Arabidopsis WT plants grown in vermiculite with and without POT1 under full P, low P, CaP, AlP, FeP and HA for two weeks. Bar graphs show combined data from fifteen independent experiments (n=15; unpaired two-tailed t test, ***P<0.001; **P<0.01; *P<0.05). Concentration of total phosphorus content in Arabidopsis shoots significantly increased after growth in POT1 presence in vermiculite soil (n=3; unpaired two-tailed t test, **P<0.01).

[0022] Figure 5 POT1 promotes growth of leafy Brassica rapa under insoluble phosphate and phosphate limiting conditions. (A) Representative images of Brassica rapa plants grown with and without POT1 under full P, low P, CaP, AlP, FeP and HA as sole P sources. Ten-day-old Brassica rapa plants were transferred to POT1 pre-inoculated and non-inoculated nutrient-poor vermiculite soil and harvested after four weeks. Scale bar represents 5 cm. Bar graphs show shoot fresh weight (B), leaf number (C), leaf area index of the 4th leaf (D) of Brassica rapa plants co-cultured with POT1 after four weeks under full P, low P, CaP, AlP, FeP and HA conditions. Bar graphs show combined data from nine independent experiments (n=9; unpaired two-tailed t test, ***P<0.001; **P<0.01; *P<0.05). (E) Shoot P content of Brassica rapa plants co-cultured with and without POT1 after four weeks. Concentration of total phosphorus content in Brassica rapa shoots significantly increased after growth in POT1 presence in vermiculite soil (n=3; unpaired two-tailed t test, **P<0.01).

[0023] Figure 6 Results of pH and nutrient analysis of soils used for plant growth experiments. (A) pH comparison of commercial soil and Lim Chu Kang (LCK) soil used for plant growth promotion experiments with POT1. (B) Bar graphs show comparison of phosphorus content in commercial soil as well as Lim Chu Kang soil. (C) Representative images of phosphate fixation at different soil pH levels.

[0024] Figure 7POT1 promotes plant growth indices of leafy vegetables in Singaporean local soils. (A) Representative images of Brassica chinensis plants grown in LCK soil with and without POT1. Ten-day-old B. chinensis plants were transferred to LCK soil pre-inoculated and uninoculated with POT1 and harvested after four weeks. Scale bar represents 5 cm. Bar graphs show shoot fresh weight (B), leaf number (C), leaf area index of the 3rd and 4th leaves (D) of B. chinensis plants co-cultured with POT1 after four weeks. Bar graphs show combined data from twelve independent experiments (n = 12; unpaired two-tailed t-test, ***P < 0.001; **P < 0.01; *P < 0.05). (E) Shoot P content of B. chinensis plants co-cultured with and without POT1 after four weeks. Concentration of total phosphorus content in B. chinensis shoots was significantly increased after growth in the presence of POT1 in LCK soil (n = 3; unpaired two-tailed t-test, **P < 0.01).

[0025] Figure 8 POT1 promotes plant growth indices of rice in Singaporean local soils. (A) Representative images of rice plants grown in LCK soil with and without POT1 after six weeks. Scale bar represents 10 cm. Fourteen-day-old rice seedlings were transferred to LCK soil pre-inoculated and uninoculated with POT1 and grown for three months. Bar graphs show plant height (B), tiller number (C), shoot P content after six weeks (D) and seed weight per plant (E) and seed number per plant (F) co-cultured with and without POT1 in LCK soil. Bar graphs show combined data from twelve independent experiments (n = 12; unpaired two-tailed t-test, ***P < 0.001; **P < 0.01; *P < 0.05).

[0026] Figure 9 Comparison of growth-promoting activities of POT1 with other Penicillium strains under P-limiting conditions is depicted. (A) Arabidopsis thaliana plants grown with POT1 and other Penicillium strains under P-free conditions. Four-day-old plants were transferred to P-free medium pre-conditioned with and without POT1, P. bilaiae, P. chrysogenum, P. janthinellum and P. simplicissimum for 10 days under in vitro conditions. Scale bar represents 1 cm. Bar graphs show primary root length (B), shoot fresh weight (C), root fresh weight (D) of A. thaliana WT plants co-cultured with P. bilaiae, P. chrysogenum, P. janthinellum and P. simplicissimum for 10 days under -P conditions. Bar graphs show combined data from three independent experiments (n = 3; unpaired two-tailed t-test, ***P < 0.001; **P < 0.01; *P < 0.05).

[0027] Figure 10 Comparison of POT1 growth promoting activity with other Penicillium strains under insoluble phosphate conditions is depicted. (A) Arabidopsis plants grown with POT1 and other Penicillium strains such as P. bilaiae, P. chrysogenum, P. simplicissimum and P. simplicissimum under full P, no P and CaP conditions. Ten-day-old plants were transferred to nutrient-poor vermiculite soil pre-inoculated with POT1, P. bilaiae, P. chrysogenum, P. simplicissimum and P. simplicissimum and un-inoculated and harvested after four weeks. Scale bar represents 1 cm. Bar graphs show shoot fresh weight (B), anthocyanin content (C) of Arabidopsis WT plants co-cultured with POT1, P. bilaiae, P. chrysogenum, P. simplicissimum and P. simplicissimum under full P, no P and CaP conditions for 10 days. Bar graphs show combined data from three independent experiments (n=3; unpaired two-tailed t-test, ***P<0.001; **P<0.01; *P<0.05).

[0028] Figure 11 Plant growth promotion of Arabidopsis by POT1 under P-sufficient and P- limiting conditions is shown (related to C and 1H). Four-day-old Arabidopsis WT, Col-0 seedlings were transferred to full P (A) and low P (B) medium pre-cultured with and without POT1. Root length was measured after ten days of co-cultivation. POT1 treatment significantly increased primary root length. Scale bar represents 1 cm. Figure 1

[0029] Figure 12 POT1 elongates primary roots of Arabidopsis in the absence of any soluble P source is shown. (A) Representative images of Arabidopsis plants grown under -P conditions with and without POT1. Four-day-old Arabidopsis plants were transferred to -P medium inoculated and un-inoculated with POT1 for 10 days under in vitro conditions. Bar graphs show primary root length (B), shoot fresh weight (C), root fresh weight (D) and shoot P content (E) of Arabidopsis WT plants co-cultured with POT1 under no P conditions. Bar graphs show combined data from ten independent experiments (n=10; unpaired two-tailed t-test, ***P<0.001; **P<0.01; *P<0.05). Scale bar represents 1 cm.

[0030] Figure 13 Representative images of Arabidopsis WT leaves grown with and without POT1 under full P, low P, CaP, AlP, FeP and HA as sole P source are depicted. Figure 13 POT1 increases leaf number of Arabidopsis WT Col-0 when cultured under insoluble phosphate sources, P-sufficient and P-limiting conditions is shown (related to Figure 4 C and 1H).

[0031] ​Figure 14 Figure 10 shows that POT1 reduces anthocyanin accumulation in Arabidopsis leaves under P-sufficient and P-limited conditions. Bar graphs represent anthocyanin content in Arabidopsis plants grown with different phosphate sources inoculated and not inoculated with POT1 in vermiculite soil. Relative anthocyanin content per gram fresh weight in (1-4) leaves (A), (5-8) leaves (B) and (9-12) leaves (C) of Arabidopsis grown with and without POT1 under full P, low P, no P, CaP, AlP, FeP and HA as sole phosphate source in three independent experiments (n=3; unpaired two-tailed t-test, ***P<0.001; **P<0.01; *P<0.05).

[0032] Figure 15 Figure 11 shows that POT1 promotes growth indices of Arabidopsis when grown in vermiculite soil supplied with any soluble phosphate source. (A) Representative images of Arabidopsis WT plants grown with and without POT1 under full P and no P conditions. Ten-day-old plants were transferred to POT1 pre-inoculated vermiculite for 14 days. Scale bar represents 1 cm. Bar graphs represent shoot fresh weight (B), leaf number (C) and leaf area index (D) of the seventh and eighth leaves of Arabidopsis WT plants grown with and without POT1 under full P and no P conditions in vermiculite. Bar graphs show combined data from fifteen independent experiments (n=15; unpaired two-tailed t-test, ***P<0.001; **P<0.01; *P<0.05). Scale bar represents 5 cm. (E) Total phosphorus content concentration in Arabidopsis shoots significantly increased after growth in the presence of POT1 without any phosphate source (n=3; unpaired two-tailed t-test, **P<0.01).

[0033] Figure 16 Figure 12 shows that POT1 promotes growth indices of leafy vegetables when grown in vermiculite supplied with any soluble phosphate. (A) Representative images of Brassica rapa grown with and without POT1 under full P and no P conditions. Ten-day-old plants were transferred to POT1 pre-inoculated and not inoculated vermiculite for seven days. Scale bar represents 5 cm. Bar graphs represent shoot fresh weight (B), leaf number (C), leaf area index (D), shoot phosphorus content (E) of the seventh and eighth leaves of Brassica rapa plants grown with and without POT1 under full P and no P conditions in vermiculite. Bar graphs show combined data from nine independent experiments (n=9; unpaired two-tailed t-test, ***P<0.001; **P<0.01; *P<0.05).

[0034] Figure 17Representative images of Brassica chinensis leaves grown in the presence and absence of POT1 under full P, low P, CaP, AlP, FeP and HA as the sole P source are depicted. Ten-day-old B. chinensis plants were grown in vermiculite pre-inoculated with POT1 for four weeks. Scale bar represents 1 cm. POT1 increased leaf number of B. chinensis plants in P-sufficient and P-limited conditions in vermiculite soil test (with Figure 5 Relevance).

[0035] Figure 18 Soil nutrient profiles of commercial soil as well as local soil in Singapore are shown. Bar graphs show the comparison of nutrient content of N, P, K, Ca, Mg, Fe (A) and Na, Cu, Mn, Zn and B (B) in commercial soil as well as Lim Chu Kang soil in triplicate independent experiments (n = 3; unpaired two-tailed t-test, ***P < 0.001; **P < 0.01; *P < 0.05).

[0036] Figure 19 Representative images of B. chinensis plants grown in the presence and absence of POT1 in Lim Chu Kang soil for four weeks are depicted.

[0037] Figure 20 Representative images of Temasek Rice (Oryza sativa var. Indica) grown in the presence and absence of POT1 in Lim Chu Kang soil for six weeks are depicted.

[0038] Figure 21 POT1 promotes leafy vegetable growth in local soil in Singapore is shown. (A) Representative images of B. chinensis plants grown in the presence of active POT1, heat-killed POT1 and absence of POT1 in Lim Chu Kang soil. Ten-day-old B. chinensis plants were grown in vermiculite pre-inoculated with POT1 for four weeks. Bar graphs show that the shoot fresh weight (B), leaf number (C), leaf area index of the 5th and 6th leaves (D) of B. chinensis plants grown with POT1 were increased after four weeks. Bar graphs show combined data from nine independent experiments (n = 9; unpaired two-tailed t-test, ***P < 0.001; **P < 0.01; *P < 0.05). Scale bar represents 5 cm.

[0039] Figure 22POT1 promotes the growth of rice in local soil in Singapore. (A) Representative images of rice grown in LCK soil with active POT1, heat-inactivated POT1, and without POT1. Two-week-old Lim Chuan See rice (Oryza sativa var. Nipponbare) seedlings were grown in LCK soil for three months. Bar graphs showing the plant height (B), tiller number after six weeks (C), total seed weight per plant (D), and seed number per plant (E) of rice co-cultured with active POT1, heat-inactivated POT1, and no POT1. Bar graphs show combined data from nine independent experiments (n = 9; unpaired two-tailed t test, ***P < 0.001; **P < 0.01; *P < 0.05). Scale bar represents 5 cm.

[0040] Figure 23 Bar graphs showing the concentration of soluble P produced by POT1, Penicillium bilaiae, P. chrysogenum, P. simplicissimum, and P. simplicissimum in modified Pikovskaya broth with CaP. Error bars show standard deviation. Each treatment included three biological replicates, each containing three technical replicates (n = 3; unpaired two-tailed t test, ***P < 0.001; **P < 0.01; *P < 0.05).

[0041] Figure 24 A agar plate containing CAS-blue agar and MEA medium inoculated with POT1. POT1 secretes an iron siderophore that changes the color of the medium from blue to purple-red, as indicated by the arrow. Figure 24 POT1 secretes an iron siderophore into the medium.

[0042] Figure 25 Effect of POT1 treatment on Arabidopsis plants under Al stress on agar plates. (A) Images of untreated Arabidopsis seedlings (control), Arabidopsis seedlings grown on 100 mM AlCl3 (Al), and Arabidopsis seedlings grown on 100 mM AlCl3 together with POT1 colonies (Al + POT1). The red line indicates the maximum root length. Comparison of primary root length (B), root fresh weight (C), shoot fresh weight (D), leaf area of the two largest leaves (E, F) of Arabidopsis seedlings grown under control, Al, and Al + POT1 conditions at pH 4 and pH 5.6. Data represent the mean ± standard deviation (SD) of 9 replicates. Statistical significance of measurements was determined by Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001).

[0043] Figure 26Effects of POT1 treatment on endogenous aluminum, nitric oxide and callose concentrations in Arabidopsis roots under Al stress. Arabidopsis roots grown under control, control + POT1, 100 μΜ AlCl3(Al) and 100 μΜ AlCl3 together with POT1 inoculation (Al + POT1) were stained with morin (A), DAF-FM DA (B) and aniline blue (C) and observed by confocal laser scanning microscopy to visualize aluminum (A), nitric oxide (B) and callose (C) contents, respectively. All images represent 9 seedlings per treatment. Bars, 100 μm.

[0044] Figure 27 Effects of POT1 inoculation on morphological parameters of Arabidopsis plants grown in soil under Al stress. (A) Images of Arabidopsis plants grown under 0, 10, 20, 30, 40, 50 and 100 mM of AlCl3. (B and C) Images of Arabidopsis plants grown under 0 (control), 50 mM AlCl3(Al) and 50 mM AlCl3 together with POT1 inoculation (Al + POT1). Scale bar: 100 mm. Comparison of shoot fresh weight (D), shoot Al content (E), total anthocyanin content (F) and total chlorophyll content (G) of Arabidopsis plants grown in soil under control, Al and Al + POT1 conditions. Data represent the mean ± SD of 6 replicates for d, 3 replicates for e and 5 replicates for f and g. Statistical significance of measurements was determined by Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001).

[0045] Figure 28 Effects of POT1 inoculation on morphological parameters of Brassica chinensis plants grown in soil under Al stress. (A) Images of Brassica chinensis plants grown under 0, 10, 20, 30, 40, 50 and 100 mM of AlCl3. (B) Images of Brassica chinensis plants grown under 0 (control), 20 mM AlCl3(Al) and 20 mM AlCl3 together with POT1 inoculation (Al + POT1). Scale bar: 100 mm. Comparison of shoot fresh weight (C) and shoot Al content (D) of Brassica chinensis plants grown in soil under control, Al and Al + POT1 conditions. Data represent the mean ± SD of 15 replicates for c; 3 replicates for d. Statistical significance of measurements was determined by Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001).

[0046] Figure 29Comparison of the effects of different Penicillium strains treatments on Arabidopsis plants under Al stress grown in agar plates. (A) Images of Arabidopsis seedlings grown on 100 mM AlCh, in the absence of Penicillium strains (control) or in the presence of the following Penicillium strains: POT1, P. bilaiae, P. chrysogenum, P. simplicissimum and P. simplicissimum. Comparison of primary root length (B), root fresh weight (C) and shoot fresh weight (D) of Arabidopsis seedlings grown under Al stress, without or with Penicillium strains. Data represent the mean of 3 replicates ± SD. Statistical significance of the measurements was determined by Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001).

[0047] Figure 30 Comparison of the effects of different Penicillium strains treatments on Arabidopsis plants under Al stress grown in soil. (A) Images of Arabidopsis plants grown on 100 mM AlCh, in the absence of Penicillium strains (control) or in the presence of the following Penicillium strains: POT1, P. bilaiae, P. chrysogenum, P. simplicissimum and P. simplicissimum. Comparison of total anthocyanins (B) and total chlorophyll content (C) of Arabidopsis plants grown under Al stress, without or with Penicillium strains. Data represent the mean of 3 replicates ± SD. Statistical significance of the measurements was determined by Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001).

[0048] Figure 31 Comparison of two POT1 treatment experimental conditions on Arabidopsis seedlings under Al stress grown in agar plates. (A) Images of Arabidopsis seedlings transferred to plates containing 0 mM AlCh (control), 100 mM AlCh (Al), 1 -week-old POT1 inoculum grown on 100 mM AlCh (Al+POT1#1) and POT1 cells just inoculated on 100 mM AlCh (Al+POT1#2). (B) Comparison of primary root length of Arabidopsis seedlings grown under control, Al, Al+POT1#1 and Al+POT1#2 conditions. Data represent the mean of 3 replicates ± standard deviation (SD). Statistical significance of the measurements was determined by Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001).

[0049] Figure 32 Effect of POT1 treatment on Al content in roots of Arabidopsis plants under Al stress. Al content in roots was measured using inductively coupled plasma-optical emission spectroscopy (ICP-OES). Data are the mean of at least 50 root measurements ± SD. Statistical significance of the measurements was determined by Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001). DETAILED DESCRIPTION

[0050] For convenience, references cited herein are listed in the Reference List at the end of the Examples section. The entire contents of all of these references are incorporated herein by reference.

[0051] Biological material preservation

[0052] The following biological materials have been deposited under the terms of the Budapest Treaty with the Agricultural Research Service Culture Collection (NRRL), 1815 University Street, North, Peoria, IL 61604, and given the following accession numbers:

[0053]

[0054] Definitions

[0055] For convenience, certain terms used in the specification, examples, and appended claims are collected here.

[0056] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present application, are those well known and commonly employed in the art. The techniques used to manipulate the cells and to manipulate the nucleic acids are well known and are described in the literature. Generally, the techniques used in the present application are well known and commonly employed in the art. In the event that any terminology is used that is not understood by a person of ordinary skill in the art, it is to be read in the context of the specification and / or the appended claims to understand the actual meaning. In addition, the following technical comments and definitions are provided. These definitions should not limit the scope of the application to only these terms, but are presented to better understand the following description.

[0057] As used herein, unless otherwise indicated or otherwise evident from context, “a” or “an” can mean one or more.

[0058] As used herein, “comprising” or “including” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. However, in the context of the present disclosure, the term “comprising” or “including” also includes “consisting of”. Variations of the word “comprising”, such as “comprise” and “comprises”, and variations of the word “including”, such as “include” and “includes”, have correspondingly varied meanings.

[0059] As used herein, the terms "an efficacious amount" and "an effective amount" are used interchangeably and refer to an amount of a fungal strain and / or a composition comprising the fungal strain sufficient to achieve a beneficial or desired result (i.e., to promote plant growth) in a statistically significant manner. For example, as used in this context, an effective amount of a composition of the present disclosure can result in a broad improvement in plant growth characteristics such as, but not limited to, improved root development, increased leaf area, increased plant yield, increased seed germination, increased photosynthesis, and / or increased biomass of the plant.

[0060] As used herein, the term "improving the quality of a growth medium" refers to increasing the amount and / or effectiveness of nutrients (such as phosphorus) in the medium that are required or beneficial for plant growth and development. In addition, the term also refers to decreasing or minimizing the amount and / or effects of elements (such as aluminum) in the medium that can be detrimental to plant growth and development. Thus, by applying the fungal strains and / or compositions of the present disclosure to improve the quality of a growth medium, plant growth in the medium is facilitated and promoted.

[0061] As used herein, the terms "low phosphorus content", "phosphorus deficiency", "phosphorus limitation", "P deficiency", and "P limitation" are used interchangeably and refer to an amount of bioavailable phosphorus in a medium in which a plant is growing that is lower than the optimal amount required for normal growth and development of the plant.

[0062] As used herein, the terms "medium" or "plant growth medium" are used interchangeably and refer, in the context of the present invention, to any substrate that can be routinely used for cultivating plants and that allows for plant growth and development.

[0063] As used herein, the terms "plant growth promotion", "plant growth enhancement", "promoting and / or enhancing plant growth", and any other equivalent grammatical meanings thereof are used interchangeably and refer to an improvement in one or more characteristics of plant growth and / or development of a treated plant as compared to a control plant (e.g., a plant treated with a composition of the present invention as compared to a control, untreated plant). It will be appreciated by those skilled in the art that examples of characteristics of plant growth / development include, but are not limited to: plant height, leaf number, leaf surface area, leaf length, leaf mass, leaf volume, photosynthesis rate, rooting rate, root area, root volume, root surface area, root length, root diameter, root mass, root number, shoot number, tiller number, shoot diameter, shoot length, shoot mass, shoot diameter, shoot surface area, shoot volume, number of seeds per plant, seed weight, seed germination, seedling development, amount of phosphorus in the roots, amount of phosphorus in the shoots, aluminum tolerance, amount of aluminum in the roots, amount of iron in the roots, amount of callose in the roots, amount of nitric oxide in the roots, amount of iron in the roots, and the like.

[0064] As used herein, the term "reducing aluminum (Al) stress / toxicity" refers to reducing the development of aluminum sensitivity symptoms and / or increasing tolerance to the presence of aluminum in a growth medium. For example, in this context, plants grown in a medium treated with the fungal strain and / or composition of the present application can show reduced symptoms of aluminum toxicity (such as reduced aluminum-mediated callose deposition in their roots) as compared to untreated control plants grown under similar environmental conditions.

[0065] As used herein, the term "phosphorus source" refers to a phosphate compound that is not fully available for uptake by plants at least under the conditions of the medium (such as soil) in question.

[0066] The following is a description of illustrative, non-limiting embodiments of the present application.

[0067] The present application is based, in part, on the identification of a novel Penicillium strain, designated Penicillium olsonii TLL1 ("POT1"), which has been found to have a higher phosphorus solubilizing capacity and the ability to reduce aluminum toxicity in plants. In this regard, the present inventors have successfully determined that inoculating a growth medium for plants with the fungal strain of the present disclosure can have a positive impact on the quality and internal environment of the growth medium, thereby enhancing the growth and development of plants.

[0068] In one aspect, disclosed herein is an isolated fungal strain of the Penicillium olsonii species, having the accession number NRRL 68252, deposited at the Agricultural Research Service Culture Collection (NRRL) on January 18, 2023.

[0069] Plant growth promoting composition

[0070] In another aspect, disclosed herein is a plant growth promoting composition comprising the isolated fungal strain of the present disclosure, its mycelium, hyphae, spores, and / or cell-free culture filtrate thereof. In some embodiments, the composition comprises an inoculum of the isolated fungal strain disclosed herein.

[0071] The compositions disclosed herein are intended to promote plant growth by altering the growth medium environment in which the plants are grown. For example, the compositions disclosed herein are capable of effectively solubilizing insoluble or slightly soluble phosphorus sources that are poorly effective and mobile in growth media, such as soil. Advantageously, the compositions disclosed herein can increase the bioavailability of phosphorus in the growth medium and promote nutrient uptake of available phosphorus to promote the growth and development of plants.

[0072] It will be appreciated that aluminum is a known plant growth inhibitor and is considered toxic to most plants. Advantageously, the compositions disclosed herein can also promote aluminum tolerance by reducing aluminum uptake and translocation into plants.

[0073] Additionally, the compositions disclosed herein can also advantageously promote root elongation by inhibiting and reducing the deposition of iron, nitric oxide, and / or callose in plant roots that can result from phosphate deficiency and / or aluminum toxicity. Thus, in some embodiments, the compositions disclosed herein can (a) increase the availability of phosphorus for uptake by plants in a medium in which the plants are grown, (b) reduce aluminum stress / toxicity in plants grown in a medium that includes aluminum, and / or (c) inhibit negative components of plant root growth, such as the accumulation of iron, nitric oxide, and / or callose in the roots.

[0074] In view of the phosphate solubilizing ability of the compositions disclosed herein, the compositions can further include an additional phosphorus source to increase the bioavailability of phosphorus uptake by plants. In some embodiments, the phosphorus source can be a soluble phosphorus compound, a slightly soluble phosphorus compound, or an insoluble phosphorus compound. Preferably, the phosphorus source is an insoluble or slightly soluble phosphorus compound. Examples of insoluble or slightly soluble phosphorus compounds can include, but are not limited to, calcium phosphate, dicalcium phosphate, tricalcium phosphate, tetracalcium phosphate, ferric phosphate, aluminum phosphate, hydroxyapatite, rock phosphate, and fluorapatite. In some embodiments, the phosphorus source is calcium phosphate, tricalcium phosphate, ferric phosphate, aluminum phosphate, and / or hydroxyapatite.

[0075] It will be appreciated by those skilled in the art that any medium conventionally used for cultivating plants is suitable for use in the present application. Examples of plant growth media can include, but are not limited to, soil (such as rice soil or upland soil), liquid medium (such as water), artificial soil, agar, vermiculite, compost, polysaccharide, peat moss, mulch, tree bark, wood fiber, logs, clay, mold, sand, gravel, peat, straw, growstones, rice hulls, brick fragments, perlite, wool, pumice, rock wool, polystyrene packing fillers, substrates suitable for hydroponics (such as expanded clay aggregates), any aqueous growth medium suitable for hydroponics, or combinations thereof. In some embodiments, the medium in which the plants are grown can be an agar medium, a liquid medium, soil, or vermiculite.

[0076] Advantageously, the compositions disclosed herein are also capable of improving the quality of the medium in which the plants are grown. Thus, it can be useful to apply the compositions of the present disclosure to plant growth media that can not be ideal / specifically advantageous for supporting plant growth. For example, the medium in which the plants are grown can be phosphate deficient, can include a low phosphorus content, and / or include insoluble or slightly soluble phosphate compounds. In another example, the medium in which the plants are grown can be acidic and / or can include aluminum.

[0077] The compositions disclosed herein can be formulated in solid, liquid, or powder form, as is known in the art. In some embodiments, the compositions can be formulated in granular form, concentrated form, or ready-to-use form. In some embodiments, the compositions can also be formulated as a seed coating.

[0078] In some embodiments, the composition can include a carrier. For example, the carrier can be an agriculturally acceptable carrier or a soil compatible carrier. In some embodiments, the carrier can include, but is not limited to, water, oil, a surfactant, an additive, a thickening agent, a dispersant, an anti-caking agent, a stabilizer, a coating, a preservative, a colorant, a polymer, or a combination thereof. It will be appreciated that one of skill in the art can readily determine an appropriate carrier that can be used taking into account factors such as the type of growth medium in which the composition is to be applied, the type of plant, the formulation of the composition (e.g., solid, liquid, or powder form), and the like.

[0079] The compositions disclosed herein can also include other ingredients that can facilitate the growth of the fungal strain in the culture medium. For example, the composition can further include inorganic salts, nutrients such as sugars (e.g., glucose and sucrose), and / or other organic materials that can serve as a substrate for the fungi of the present disclosure.

[0080] The amount of the composition used in the present disclosure is not limited by any particular aspect. The amount of the composition to be used can depend on a variety of factors such as the type of plant species being treated, the type of growth medium, and the volume of the growth medium in which the composition is to be applied, the formulation of the composition, the climatic conditions, and the like. For any given situation, an effective amount of the composition disclosed herein should be applied, and can be readily determined by one of ordinary skill in the art through simple routine experimentation. In this regard, an effective amount is the amount necessary to establish sufficient fungal growth such that plant growth is improved in the presence of the composition. It can be appreciated that if an effective amount is used, a positive, observable effect on plant growth will result. Likewise, if an insufficient amount is applied, no positive effect can be observed. For example, the amount of the composition to be applied can include about 0.001-1.0 Kg of fungal spores and mycelium (fresh weight) per hectare, or about 1.0 x 10 2 colony forming units (CFU) / mL to about 5.0 x 10 12 CFU / mL. In some embodiments, the composition can include at least 0.1 g of mycelium. In some embodiments, the composition can include at least 0.5 g of mycelium. In some embodiments, the composition to be applied can include at least 0.5 g of mycelium per pot of plants.

[0081] In another aspect, a plant seed is provided that is coated with the isolated fungal strain of the present disclosure or the composition disclosed herein. In some embodiments, the plant seed is further coated with a soil compatible carrier. For example, the amount of the composition to be applied can include about 1.0 x 10 2 -1.0 x 10 7 CFU per seed. Methods of preparing seed coating formulations are known in the art.

[0082] In another aspect, there is provided a composition of the present disclosure for use as a fertilizer. In another aspect, there is also provided the use of a composition disclosed herein as a fertilizer. In some embodiments, the fertilizer can be formulated as a liquid, powder, or granular formulation. In some embodiments, the fertilizer can be formulated as a soluble or slow-release formulation. In some embodiments, the fertilizer can be formulated as a dry biomass (such as a dust or granule) or a suspension (such as a water-based or emulsion). It will be appreciated that methods of formulating fertilizers are known in the art.

[0083] Method for promoting plant growth

[0084] Also disclosed herein are methods of promoting plant growth. In one aspect, there is provided a method of promoting plant growth, the method comprising applying an effective amount of a growth promoting composition of the present disclosure to a medium for plant growth, wherein the application of the composition promotes plant growth as compared to a plant grown in an untreated medium.

[0085] Advantageously, the methods disclosed herein can promote plant growth by actively altering the growth medium environment and improving the quality of the medium for plant growth. In some embodiments, the application of the composition in the methods disclosed herein can (a) increase the phosphorus availability for plant uptake in a medium for plant growth, (b) reduce aluminum (Al) stress / toxicity in plants grown in a medium comprising aluminum, and / or (c) inhibit negative components of root growth, such as the accumulation of iron, nitric oxide, and / or callose in roots.

[0086] In some embodiments, the methods disclosed herein can be applied to a medium comprising at least one of: low phosphorus content, insoluble phosphate, or aluminum. In some embodiments, the medium can be an agar medium, a liquid medium, soil, or vermiculite. In some embodiments, the soil can be a paddy soil or an upland soil.

[0087] For the purposes of the compositions and methods of the present disclosure, the target plant(s) are not particularly limited. For example, the plant can be a dicot or a monocot. Examples of plants can include, but are not limited to, Gramineae plants (e.g., barley, corn, wheat, sorghum, sugarcane, and rice species such as Oryza sativa or Oryza sativa var. indica), Leguminosae plants (e.g., soybean, fava bean, and pea); Brassicaceae plants (e.g., broccoli, cauliflower, cabbage, Chinese cabbage, Japanese radish, turnip, Arabidopsis species such as Arabidopsis arenosa, Arabidopsis lyrata, Arabidopsis thaliana, wild-type Arabidopsis (Col-0)), Brassica species such as Brassica chinensis komatsuna, Brassica campestris, Brassica rapa var. nipposinica, Brassica juncea, and Brassica rapa var. chinensis (Chinese cabbage)), Solanaceae plants (e.g., tobacco, eggplant, tomato, sweet pepper, and hot pepper), Chenopodiaceae plants (e.g., beet and spinach), and Asteraceae plants (e.g., lettuce and butter lettuce). In some embodiments, the plant can be a Brassica species, an Arabidopsis species, or an Oryza species. In some embodiments, the plant can be Chinese cabbage, wild-type Arabidopsis (Col-0), or Oryza sativa var. indica.

[0088] Unless otherwise indicated or understood from the context and understanding by those of ordinary skill in the art, values expressed in a range can assume any specific value or sub-range within that range in various embodiments up to one-tenth of the lower limit of the range, unless the context clearly dictates otherwise. Reference to a numerical value is generally intended to refer to a range of values falling within ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5% of that value, unless otherwise indicated or understood from the context. In any embodiment in which a numerical value is preceded by "about," an embodiment in which the precise value is recited is provided. In embodiments in which a numerical value is provided that is not preceded by "about," an embodiment in which the value is preceded by "about" is also provided. In ranges preceded by "about," unless the context clearly dictates otherwise, "about" applies to both the lower limit and the upper limit of the range, or to the lower limit or the upper limit, in embodiments in which provided. Where a phrase such as "at least," "up to," "no more than," or the like precedes a list of numbers, unless the context clearly dictates otherwise, the phrase is understood to apply to each number in the list in various embodiments (with the understanding that 100% of a value, e.g., a value expressed in percentage, can be the upper limit, depending on the context). For example, "at least 1, 2, or 3" is understood in various embodiments to be "at least 1, at least 2, or at least 3." It is also understood that any and all reasonable sub- and upper-limits are expressly contemplated.

[0089] Having now generally described the application, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting of the present application.

[0090] Examples

[0091] Example 1 : Phosphate solubilizing fungus (PSF) Penicillium olivascens TLL1 promotes plant growth and increases phosphate use efficiency

[0092] Methods and Materials

[0093] Identification and genotyping of PSF strains

[0094] PSF was isolated by inoculating environmental samples onto Pikovskaya ("PVK") agar containing 0.5% tribasic calcium phosphate (CaP), pH 6.8-7.2, and incubating at 25°C for 7 days. A clear halo of insoluble phosphate around the fungal colonies indicated difficulty in dissolving the phosphate. The ratio of colony diameter to the insoluble phosphate halo was calculated to determine the rate of phosphate solubilization.

[0095] Total genomic DNA was extracted from PSF strains using the CTAB method. One hundred nanograms of genomic DNA from PSF strains was used in a high-fidelity Phusion Hot Start II PCR Master Mix (New England Biolabs, Ipswich, MA) with primers designed to amplify the 18S rRNA gene region. TMDNA polymerase to amplify seven loci such as Internal Transcribed Spacer (ITS), beta-tubulin (benA), translation elongation factor-1-alpha (TEF1a), calmodulin (CaM), 28S ribosomal large subunit (LSU), 18S ribosomal small subunit (SSU) and mini-chromosome maintenance protein (MCM7). Genotyping analysis was performed using primers shown in Table 1. PCR reaction profile consisted of an initial denaturation at 98°C for 3 minutes; followed by 35 cycles of 98°C for 10 seconds, 55-68°C for 30 seconds and 72°C for 1 minute; and a final extension at 72°C for 5 minutes. Sequences of PCR amplicons were determined using Sanger sequencing and contigs were assembled using Bioedit tool software and analyzed by BLAST search at NCBI.

[0096] Table 1: Oligonucleotide primers used for genotyping.

[0097]

[0098] Phylogenetic trees were constructed for each locus separately using the Maximum Likelihood (ML) algorithm with 1000 bootstrap replicates using the Tamura Nei model using MEGA v 7.0 software. Reference sequences of Penicillium genes were also used for alignment. The final presented trees show bootstrap cutoff level higher than 65% (Visagie et al., 2014). The identified PSF strain was named Penicillium olsonii TLL1 (POT1).

[0099] Plant material and growth conditions

[0100] For seedling growth, Arabidopsis WT (Col-0), Pakchoi (Brassica rapachinensis) and rice (Oryza sativa; Singapore plant variety PVP / 14 / 00003Z, rice variety indica) seeds were germinated on medium containing Murashige and Skoog salts, 0.25 mM MES, 10 g / l sucrose and 0.8% agar.

[0101] In vitro growth test of Arabidopsis ecotype Col-0 was evaluated under full P, low P and -P conditions (0.50 mM, 0.01 mM and 0 mM KH2PO4, respectively) in modified Hoagland solution at pH 5.6. Four-day-old Arabidopsis seedlings were transferred to test medium pre-incubated with POT1 inoculum. Root length of seedlings was estimated after 10 days using Image J software (https: / / imagej.nih.gov / ij / download).

[0102] For soil tests, vermiculite was used as a non-nutrient soil for studying plant growth under insoluble phosphate conditions. It was washed three times with deionized water to remove any soluble P and air-dried at room temperature. Plants were grown at 22°C (Arabidopsis) and 25°C (Brassica) under 75% relative humidity.

[0103] Plants were irrigated twice a week with Hoagland's solution containing full P (0.5 mM KH2PO4), low P (0.01 mM KH2PO4), -P (0 mM KH2PO4), 0.5 mM tricalcium phosphate (0.5 mM CaP), 0.5 mM aluminum phosphate (0.5 mM AlP), 0.5 mM ferric phosphate (0.5 mM FeP), and 0.5 mM hydroxyapatite (0.5 mM HA).

[0104] To test plant growth by P-olytic activity, POT1 mycelium (0.5 g) was watered in vermiculite for 7 days prior to transferring seedlings. Seven-day-old Arabidopsis and Brassica seedlings were transferred to vermiculite and subjected to different phosphate sources.

[0105] Tests were performed in vermiculite soil medium to compare POT1 with other Penicillium strains, such as P. bilaiae (ATCC 20851), P. chrysogenum (NBRC 4626), P. janthinellum (NBRC 31133), and P. simplicissimum (NBRC 106922) for CaP-olytic ability. Fungal mycelium (0.5 g) was watered in vermiculite for 7 days prior to transferring Arabidopsis seedlings and plants were irrigated twice a week with Hoagland's solution containing tricalcium phosphate as the sole P source.

[0106] Insoluble phosphate solubilizing activity analysis of POT1

[0107] POT1 was grown in the dark for two days in Prune agar medium (40 ml / L prune juice, 1 g / L yeast extract, 2.5 g / L lactose, 2.5 g / L sucrose, 20 g / L agar, pH 6.5) and for three days under light for sporulation. PSF conidia suspensions (10 8Spores / ml) were inoculated into 100 ml of modified Pikovskaya broth (containing 1 g / L of poorly soluble inorganic phosphate salts such as tribasic calcium phosphate (pH 7.0), ferric phosphate (pH 2.5), aluminum phosphate (pH 4.0), and hydroxyapatite (pH 7.0)) at 25 °C with 120 rpm for 14 days. Uninoculated media with different P sources were also retained as controls. Cultures were centrifuged at 5000 rpm for 15 min, and the supernatant was filtered with a 0.22 pm syringe filter. Soluble phosphorus content was determined using the phosphomolybdenum spectrophotometric method (Ames, 1966). POT1 was also compared with the commercial biofertilizer strain Penicillium bilaiae (ATCC 20851) for their insoluble phosphate solubilizing activity on tribasic calcium phosphate, ferric phosphate, aluminum phosphate, and hydroxyapatite. POT1 was also compared with other Penicillium strains (such as P. chrysogenum (NBRC 4626), P. purpurescens (NBRC 31133), and P. simplicissimum (NBRC 106922)) for their phosphorus solubilizing activity in CaP-modified Pikovskaya broth. The phosphorus solubilization rate was calculated using the following formula described by Qiao et al., 2019: ([soluble phosphorus content of inoculated group - soluble phosphorus content of control group] / inorganic phosphorus content of each experimental group * 100).

[0108] Effect of POT1 under local soil conditions

[0109] Growth tests of Pakchoi and Temasek Rice were performed on soils collected from Singapore Lim Chu Kang (N 1°26’7.3284”, E 103°42’48.456”) under greenhouse conditions. Seven days before the transfer of Pakchoi and Rice seedlings, POT1 mycelium including heat-inactivated POT1 (0.5 g per pot) was drenched in Lim Chu Kang soil.

[0110] Total phosphorous content analysis

[0111] Roots and shoots of Arabidopsis plants after growth in different phosphorus sources were collected. Total phosphorus content was measured by digesting 45 mg of leaf or root tissue in 1 mL 3:1 HNO3 / HCl in a microwave oven at 240 °C for 15 min, and after digestion, ten-fold dilution with water. Phosphorus content was determined using inductively coupled plasma-atomic emission spectroscopy (ICP-OES).

[0112] Callose staining

[0113] Arabidopsis plants grown under full P and low P conditions were incubated in 150 mM K2HPO4 and 0.01% aniline blue for 2 hours in the dark. Callose deposits were quantified under a fluorescence microscope using a DAPI filter (excitation 370 nm and emission 509 nm, Olympus FV 3000 (20X objective)).

[0114] NO staining

[0115] Roots were excised from Arabidopsis plants grown for 10 days in full P and low P media. Roots were treated with 10 mM DAF-FM DA (3-amino, 4-aminomethyl-2',7'-difluorofluorescein diacetate) for 15 minutes and the samples were washed with HEPES buffer (pH 4.0) for 10 minutes. Fluorescence was observed with a fluorescence microscope (excitation 495 nm and emission 515 nm, Olympus FV 3000 (20X objective)).

[0116] Anthocyanin glycoside measurement

[0117] Anthocyanin content in Arabidopsis plant leaves was determined following the protocol of Laby et al., 2000. Leaves from 14-day-old Arabidopsis plants co-cultivated with and without POT1 were divided into three groups (leaves 1-4, 5-8, 9-12). Leaves were extracted with 99: 1 methanol: HC1 (v / v) overnight at 4°C. Anthocyanin content was determined by taking the OD at 530 and 657 nm and calculated using the following formula: OD 530 - (0.25 * OD 657 )* extraction volume (ml) * 1 / tissue weight.

[0118] Total macro / micro nutrient content analysis in Lingcogang soil

[0119] Soil nutrient analysis was performed to determine the available nutrients in the Lim Chu Kang soil and commercial soil used for the cultivation of Pakchoi and rice. Nitrogen, phosphorus, potassium, calcium, magnesium, sodium, copper, manganese, zinc and boron in 1 kg of commercial soil and local soil collected from Lim Chu Kang, Singapore were analysed using Mehlich 3 extraction (Mehlich, 1984) followed by ICP-OES analysis at the National Parks Board, Singapore. 10 g of soil was mixed with 20 ml of sterile distilled water and agitated with a magnetic stirrer for 1 hour. The pH of the soil mixture was then recorded.

[0120] Siderophore determination

[0121] POT1 siderophore secretion was analyzed using a modified CAS-agar universal assay by adding CAS-blue agar on one half of the Petri dish and malt extract agar on the other half (Milagres et al., 1999). POT1 conidial suspension (10 8 spores / ml) was inoculated in the center of the medium and incubated at 25°C for 14 days to observe siderophore production. A positive reaction results in a color change of the reagent (from blue to orange, purple or magenta).

[0122] Results

[0123] Identification and genotyping of POT1

[0124] The phylogenetic relationship of POT1 with representative fungal strains in a maximum likelihood tree was constructed using seven barcode markers such as ITS, benA, TEF1a, SSU, LSU, CaM and MCM7. All DNA barcode marker sequences of POT1 strain clustered with Penicillium olsonii sequences and the strain was identified as Penicillium olsonii Figure 1 A). Figure 1 B shows the morphology of POT1 in MEA and PDA.

[0125] POT1 promotes plant root growth under P sufficient and deficient conditions

[0126] Penicillium olsonii TLL1 (POT1) mediated Arabidopsis growth promotion was tested under P- sufficient and P-deficient medium conditions Figure 1 C and H). Figure 11 Whole plates showing Figure 1 C and Figure 1 H of Arabidopsis growth test. Under P-sufficient conditions, POT1 inoculated Arabidopsis plants showed 1.2-fold longer primary root length compared to non-inoculated plants Figure 1 C and D), but there was no significant increase in shoot, root fresh weight and shoot P content Figure 1 E, F and G) of the inoculated plants compared to non-inoculated plants. Under P-deficient conditions, POT1 inoculated plants showed 2-fold longer primary root length Figure 1 ), and there was a significant 1.5-fold and 2-fold increase in shoot and root fresh weight, respectively Figure 1 J and K) of the inoculated plants compared to non-inoculated plants. Arabidopsis growth by POT1 inoculation under P-free conditions was also tested Figure 12 A). Despite the absence of phosphate source in the medium, plants co-cultured with POT1 showed 2-fold longer primary root length and 2-fold increase in root fresh weight Figure 12 B and D) of the inoculated plants compared to non-inoculated plants, while there was no difference in shoot fresh weight between inoculated and non-inoculated plants Figure 12C). Compared with uninoculated plants, the aboveground phosphate content of plants inoculated with POT1 under -P conditions increased by 1.5 times ( Figure 12 E).

[0127] POT1 inhibits callose deposition and NO accumulation

[0128] To confirm the negative component of POT1's inhibition of root growth, we investigated the effects of callose deposition and nitric oxide (NO) accumulation, as well as POT1 inoculation, under phosphate-sufficient or deficient conditions. Figure 2 Under phosphate deficiency, extensive callose formation occurs primarily in the root apical meristem, while no additional callose deposition was observed with POT1 inoculation, similar to phosphate-sufficient conditions with / without POT1. Figure 2 A). Furthermore, since NO is a downstream signaling molecule in root development under phosphorus deficiency, we used the fluorescent probe DAF-FM DA to test the ability of POT1 to inhibit endogenous NO accumulation in roots. Green fluorescence was clearly observed under phosphate-deficient conditions, indicating phosphate-dependent NO production. In contrast, NO levels in roots decreased after POT1 inoculation, suggesting that POT1 participates in root development under phosphate deficiency. Figure 2 B).

[0129] Quantitative determination of phosphate solubilizing activity of POT1

[0130] When POT1 was grown for 7 days in PVK and NBRIP media modified with Ca3(PO4)2 at 25°C, the strain produced visible halos. We measured the colony diameter (d) and the circular band of soluble phosphate (D), and calculated the phosphate solubility index based on the d / D ratio of PSF isolated from environmental samples. The d / D ratios of the PSF strain were 1.161 and 1.212 on Pikovskaya and NBRIP media, respectively. Figure 3 A, B).

[0131] POT1 contains dissolved tricalcium phosphate [Ca3(PO4)2] and hydroxyapatite [Ca2] 10 [(PO4)6(OH)2], aluminum phosphate (AlPO4) and iron phosphate (FePO4·4H2O) accompanied by a decrease in pH of the supernatant of the modified PVK medium. Figure 3 POT1 exhibited the highest phosphorus-solubilizing activity in media containing Ca3(PO4)2, followed by hydroxyapatite, aluminum phosphate, and iron phosphate. Figure 3C). Soluble phosphorus concentrations reached 2.95 mg / ml in CaP and 1.574 mg / ml in hydroxyapatite. 14 days post-inoculation, POT1 also dissolved AlPO4 and FePO4 to release 0.924 mg / ml and 0.436 mg / ml of soluble phosphorus, respectively. Figure 3 D). The phosphorus solubility of CaP is 57%, hydroxyapatite is 31%, AlP is 20%, and FeP is 3%.

[0132] POT1 produces siderophores

[0133] Siderophores are iron chelating ligands produced by plant growth-promoting rhizosphere bacteria (PGPR) and fungi, which form soluble Fe... 3+ This facilitates the dissolution and transport of iron. To investigate the possibility of POT1-generated siderophores inhibiting iron accumulation in root tip meristems, POT1 was grown on a modified Casc-agar-MEA medium, and its color changes were observed. Figure 24 The secreted siderophores were confirmed by the formation of a purplish-red color in the culture medium.

[0134] POT1 growth test in Arabidopsis and leafy vegetables under insoluble phosphate conditions

[0135] To investigate the phosphorus-solubilizing effect of POT1 on Arabidopsis thaliana under P-sufficient and P-deficient conditions (including insoluble P sources), Arabidopsis thaliana plants were co-cultured in vermiculite (a nutrient-free soil). Figure 4 Without inoculation with POT1, Arabidopsis plants grown under low P and insoluble P sources (such as tricalcium phosphate (CaP), aluminum phosphate (AlP), iron phosphate (FeP), and hydroxyapatite (HA)) showed developmental inhibition. Figure 4 A). Consistent with the growth characteristics of Arabidopsis thaliana, the aboveground fresh weight, leaf index, number of leaves, and aboveground phosphate content were all higher than those of plants not inoculated with POT1. Figure 4 B, C, D, E and Figure 13 Anthocyanin accumulation is a typical phenotype in plants growing under phosphate deficiency. Uninoculated plants showed higher anthocyanin accumulation, while inoculated plants showed lower anthocyanin accumulation than uninoculated plants. Figure 14 AC).

[0136] Under P-deficient conditions using vermiculite soil, Arabidopsis plants showed significantly increased aboveground fresh weight, leaf number, and leaf area index compared to uninoculated plants. Figure 15 BD). However, plants grown under -P conditions still showed growth defects compared to phosphate-sufficient plants. It was also examined whether POT1 colonization could also promote the growth of leafy vegetables (such as bok choy (long-stemmed Chinese cabbage)) by growing them in vermiculite under different phosphate sources.Figure 5 A). Four weeks later, the growth of Brassica chinensis plants grown in low P, tricalcium phosphate (CaP), aluminum phosphate (AlP), iron phosphate (FeP), and hydroxyapatite (HA) conditions without POT1 appeared to be stunted, while the biomass of POT1-inoculated plants increased, as shown by shoot fresh weight, leaf number, and leaf area index Figure 5 B-D and Figure 16 ). In addition, the total phosphate content in all inoculated plants was higher under insoluble phosphate conditions as well as low phosphate conditions compared to non-inoculated Brassica chinensis Figure 5 E).

[0137] When simulating-P conditions in vermiculite soil, the shoot fresh weight, leaf number, and leaf area index of plants treated with POT1 increased compared to non-inoculated plants Figure 16 ).

[0138] Growth test by PSF under local soil conditions

[0139] It was investigated whether POT1 could function in local soil in Singapore, Lim Chu Kang, to improve the growth of Brassica chinensis and rice. First, the pH and macronutrient / micronutrient content of the local soil were analyzed. The results were that the pH of the Lim Chu Kang soil was 6.5, while the pH of the commercial soil was 5.7, which is within the range of highest effectiveness of phosphate Figure 6 A and C). Figure 6 B shows the phosphate content in the commercial soil and the Lim Chu Kang soil. The phosphate content of the commercial soil was 4 times higher than that of the Lim Chu Kang soil. However, the 50 mg / kg phosphate content in the Lim Chu Kang soil meant that there was still enough condition in the soil for plants to grow (Philip et al., 2021). In addition, other macronutrients / micronutrients in the commercial soil and the Lim Chu Kang soil were also analyzed, such as nitrogen, potassium, calcium, magnesium, iron, sodium, copper, manganese zinc, and boron Figure 18 A). Notably, the content of copper, manganese, and zinc in the Lim Chu Kang soil was higher than that in the commercial soil Figure 18 B).

[0140] Brassica chinensis plants were grown in the Lim Chu Kang soil with / without POT1 for four weeks Figure 19 ). Figure 7 It was shown that POT1 inoculation improved the growth condition of Brassica chinensis plants, as their shoot fresh weight, leaf number, and leaf index were higher than those of plants without POT1 inoculation Figure 7 A-D). Consistent with the Brassica chinensis growth index, the total P content in the leaves was higher than that of plants without POT1 inoculation Figure 7 E). In addition, it was confirmed that POT1 had phosphorus solubilizing activity in monocotyledonous plants, rice Figure 8 , Figure 20The plant height of rice plants co-cultured with POT1 was significantly greater than that of plants grown without POT1 inoculation. Figure 8 A, B), number of tillers ( Figure 8 C) and aboveground P content ( Figure 8 D) All increased. Consistent with the increase in tiller number, POT1 inoculation increased the number of seeds ( Figure 8 E) and seed dry weight ( Figure 8 F).

[0141] In addition, the effects of heat-inactivated POT1 (as a negative control) on plant growth in Chinese cabbage and rice grown under Lincuogang soil conditions were tested. Figure 21 , Figure 22 Compared with live POT1 inoculation, the aboveground fresh weight, leaf number, and leaf index of pak choy inoculated with heat-inactivated POT1 all decreased. Figure 21 AD). Similarly, in rice growth tests, heat-inactivated POT1 had no effect compared to active POT1, resulting in shorter plant height, fewer tillers, and reduced seed weight and number. Figure 22 These results indicate that POT1's phosphorus-solubilizing ability helps plants absorb phosphate sources from soils containing insoluble phosphates.

[0142] Comparison of phosphate solubilizing activity and Arabidopsis growth of five Penicillium strains

[0143] The tricalcium phosphate solubility of POT1 was compared with that of other Penicillium strains (such as Penicillium chrysogenum, Penicillium micropurpurum, and Penicillium scabra). Figure 23 In CaP, the soluble phosphate concentrations in the culture supernatants of *Penicillium chrysogenum*, *Penicillium micropurpurum*, and *Penicillium scabra* were 1.69 mg / ml, 1.84 mg / ml, and 1.97 mg / ml, respectively. *Penicillium baicaleum* obtained the highest CaP solubility (3.246 mg / ml), followed by POT1 (2.96 mg / ml).

[0144] The growth-promoting activity of POT1 was also compared with that of other Penicillium strains (such as Penicillium baicalensis, Penicillium chrysogenum, Penicillium micropurpurum, and Penicillium scabra) under P-deficient conditions. Figure 9 All strains showed longer primary root lengths compared to the control group that was not inoculated with any fungus. However, when compared among the five Penicillium strains, POT1 inoculated with the longest primary root lengths (…). Figure 9 B). Similarly, the aboveground fresh weight of POT1 grafted plants (2 times) was at its highest level ( Figure 9 C). Surprisingly, there was no difference in the aboveground fresh weight between plants inoculated with *Penicillium spp.* and the control plants. Furthermore, in the case of plants co-cultured with *Penicillium micropurpurum*, a reduction in aboveground fresh weight to 1 / 2.56 ( Figure 9C). All fungal strains inoculated roots had higher fresh weight than non-inoculated plants, and the P. olivaceum strain had the highest root fresh weight Figure 9 D).

[0145] To investigate the effect of each fungus's P-solubilizing activity on plant growth, Arabidopsis growth in vermiculite was tested using an insoluble phosphate source, calcium phosphate (CaP) Figure 10 A). Figure 10 B shows that under insoluble phosphate conditions, POT1-inoculated aerial fresh weight was higher than other strains. In addition, anthocyanin accumulation, a typical phosphate deficiency response in plants, was also measured. It was shown that POT1-inoculated plants and P. simplicissimum had similar less anthocyanin accumulation, but POT1 produced higher aerial fresh weight than P. simplicissimum Figure 10 C).

[0146] Example 2: Penicillium olivascens TLL1 strain (POT1) alleviates aluminum stress in plants.

[0147] Materials and Methods

[0148] Fungal isolation and identification

[0149] Penicillium olivaceum TLL1 (POT1) was isolated and identified according to Example 1.

[0150] Plant material and germination

[0151] Seeds of wild-type Arabidopsis (Col-0) and Brassica rapa (long-petioled Chinese cabbage) were grown on 1.2% (w / v) and 0.8% (w / v) agar medium containing Murashige and Skoog (MS) basal salts, 1% (w / v) sucrose, and 2.5 mM MES (pH 5.6), respectively. After imbibition for 2 days at 4°C in the dark, seedlings were germinated in a tissue culture room at 22°C and 60% relative humidity under 16 h light / 8 h dark conditions (100 μιηοΙ m -2 -1 Light intensity).

[0152] POT1 inoculated Arabidopsis growth test under high Al conditions

[0153] ​Five-day-old Arabidopsis seedlings were transferred to modified Hoagland nutrient media at pH 4 and pH 5.6 containing 1.5% (w / v) Phytagar and treated as follows: (1) control, (2) Al (100 μΜ AlCl3) and (3) Al + POT1 (POT1 was inoculated onto Hoagland media plates containing 100 μΜ AlCl3and grown for 1 week). Modified Hoagland nutrients contained 2.5 mM Ca(NO3)2, 2.5 mM KNO3, 1 mM MgSO4, 0.75 μΜ NaFeEDTA, 0.25 mM KH2PO4, 50 μΜ H3BO3, 0.075 μΜ, NH4Mo7O 24 4, 2 μΜ ZnSO4, 10 μΜ MnCl2, 1.5 μΜ CuSO4, 0.2 μΜ CoCl2, and 0.5 mM MES. All chemicals were obtained from Sigma-Aldrich. Transferred seedlings were grown vertically for 10 days. Four additional Penicillium species (P. bilaiae (ATCC 20851), P. chrysogenum (NBRC 4626), P. janthinellum (NBRC 31133), and P. simplicissimum (106922)) were used for comparative studies.

[0154] POT1 inoculated plant growth test in Al containing soil

[0155] Two-week-old Arabidopsis and one-week-old Brassica rapa seedlings were transferred from germination media to soil. For POT1 induction treatment, 0.5 g of fresh growing and rinsed POT1 mycelium was inoculated into each soil pot one week prior to seedling transfer. Plants were grown for four weeks in a growth chamber at 23°C and ~60% relative humidity under 16 h light / 8 h dark conditions (100 μιηοΙ m -2 s -1 of light intensity). All plants were watered twice per week. Control plants were watered with tap water, while Al-treated Arabidopsis and B. rapa plants were watered with tap water containing 50 mM and 20 mM AlCl3, respectively.

[0156] Total anthocyanin and chlorophyll content measurement

[0157] Anthocyanin levels were measured as previously described by Rabino and Mancinelli, 1986. Fifty mg of Arabidopsis aerial parts were ground in liquid N2and extracted with 500 μΐ of methanol-HCl (99: 1) overnight at 4°C. Anthocyanin levels (mg / g FW) were calculated using the following formula: (absorbance (530nm) - (0.25 x absorbance (657nm) )) x extraction volume (ml) x (1 / sample weight (g)).

[0158] Chlorophyll levels were measured as previously described by Porra et al., 1989. Chlorophyll extraction was similar to anthocyanin extraction except that 5 ml of 80% acetone was used as extraction buffer. Total chlorophyll levels (mg / g FW) were calculated using the following formula: ((7.34 x absorbance (663nm) )+(17.76 x absorbance (646nm) )) / 10.

[0159] Al staining

[0160] Arabidopsis seedlings grown under control, Al and Al+POT1 conditions were excised, washed with deionized water for 10 min and stained with 100 mM morin in 10 mM MES buffer (pH 5.5) for 30 min. Then, they were washed twice with the same MES buffer and deionized water for 5 min each. Stained root tips were mounted in 15% glycerol and observed with eGFP epifluorescence microscopy (ZEISS AXIOPLAN 2).

[0161] Al content measurement

[0162] To measure total Al content, Arabidopsis shoots and roots were dried at 60°C and ground into fine powder. To 100 mg of ground powder, 5 mL of HNO3 (68%) and 1 mL of HC1 (37%) were added and incubated at 220°C for 2 h using a graphite digestion device (DigestLinc ST60D). Then, digested samples were diluted to 10 ml with deionized water and analyzed for Al content using Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) (Agilent 720) with a detection limit of 0.02 ppm.

[0163] NO detection

[0164] Nitric oxide (NO) in situ measurements were performed as described by Park et al., 2011. Root tips were stained with 5 mM DAF-FM DA (3-amino, 4-aminomethyl-2',7'-difluorofluorescein diacetate (fluorescent NO indicator dye) in 20 mM HEPES buffer (pH 7.4) for 15 min. Then, stained root tips were washed with 20 mM HEPES buffer (pH 7.4) for 10 min and observed for fluorescence under confocal laser scanning microscopy (CLSM) (excitation 495 nm, emission 515 nm; Olympus FV3000).

[0165] Callose staining

[0166] Callose staining was performed as described in Clay et al., 2009 with modifications. Root tips were washed in 150 mM K2HPO4for 10 min and stained with 150 mM K2HPO4and 0.01% (w / v) aniline blue for 1 h in the dark. Callose deposition was visualized using CLSM (excitation 370 nm, emission 509 nm; Olympus FV3000).

[0167] Results

[0168] POT1 reduces Al stress in Arabidopsis

[0169] To assess the plant growth promoting effect of Al-tolerant POT1, 5-day-old Arabidopsis seedlings were transferred to agar plates containing 100 mM Al and 100 mM Al + POT1 at pH 4 and pH 5.6 and grown for 7 days. From previous studies, it is known that seedlings grown on pH 4-AlCl3plates exhibit severe inhibition of primary root growth compared to untreated control plants Figure 25 A, B). However, when seedlings were grown on agar plates containing 100 mM Al + POT1, the primary root length was significantly higher than Al-treated plants. At pH 5.6, Al treatment did not have a significant effect on primary root growth compared to pH 4. Notably, the primary root length of POT1-treated seedlings was higher than control and Al-treated seedlings at both pH 4 and pH 5.6, indicating that POT1 not only overcame the root growth inhibition caused by Al treatment but also had an intrinsic root growth promoting effect Figure 25 A, B). Fresh weight of primary roots correlated with primary root length Figure 25 C). In addition, plants treated with POT1 also had higher shoot fresh weight and leaf area Figure 25 D-F). Figure 31 The effect of POT1 under two different experimental conditions in Arabidopsis is shown. In the first experiment, Arabidopsis seedlings were transferred to solid medium plates in which POT1 had been grown for one week, while in the second experiment, POT1 and Arabidopsis seedlings were added to solid medium plates back-to-back on the same day. Using these two experimental conditions, we not only confirmed again that plants inoculated with POT1 had longer primary roots than non-inoculated plants, but we also realized that pre-treatment of the growth medium with POT1 was not necessary to alleviate Al stress in plants.

[0170] POT1 colonizes plant roots and reduces Al, NO and callose accumulation at root tips.

[0171] To understand the effect of POT1 on Al, NO and callose accumulation, roots of Arabidopsis plants grown with 100 mM Al and 100 mM Al + POT1 were observed under confocal microscope and bright field microscope after different staining methods. Figure 26 In the control, no Al, NO and callose were detected, or only weak diffuse signals were observed, while in the roots exposed to aluminum, high levels of Al, NO and callose specific signals were observed. Interestingly, in the Al + POT1 treated plants, a drastic decrease in the levels of Al, NO and callose could be detected. When Al content was measured using Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES), the Al content of the Al + POT1 treated plants was indeed much lower than that of the Al treated plants Figure 32 A). These results indicate that POT1 successfully mitigated the accumulation of Al, NO and callose in the root elongation zone and / or root tip, thus preventing the inhibition of primary root growth.

[0172] Effect of POT1 on Arabidopsis and Brassica chinensis growth in high Al containing soil

[0173] To test the effect of aluminum toxicity in Arabidopsis and Brassica chinensis, 1-week-old seedlings were transferred to soil and treated with different concentrations (0, 10, 20, 30, 40, 50 and 100 mM) of AlCl3solution, once a week. As the Al concentration in the soil increased, Arabidopsis plants became smaller and smaller, but at 100 mM Al, plant growth was significantly retarded and they presented a purple color, which indicates anthocyanin accumulation Figure 27 A). Increasing Al concentration had a similar effect on Brassica chinensis plants Figure 28 A). Based on these results, 50 mM and 20 mM AlCl3were chosen as the optimal concentrations to study the effect of POT1 on Al toxicity in Arabidopsis and Brassica chinensis plants, respectively. While Arabidopsis plants treated with 50 mM Al + POT1 were smaller than the untreated control plants, they were significantly larger than the plants treated with Al only Figure 27 B). Al-treated plants were not only much shorter than the Al + POT1 inoculated plants, but they also senesced earlier Figure 27 C). The aerial fresh weight of the Al-treated plants was up to 1 / 4 lower than that of the control plants (Figure 38D). Although the aerial fresh weight of the Al + POT1 treated plants was slightly lower than that of the control plants, they were 3 times higher than the aerial fresh weight of the Al-treated plants Figure 27 D), which indicates that Al stress was less in the POT1 inoculated plants Figure 27 E). Not surprisingly, under 50 mM Al treatment, POT1 inoculated plants showed less stress phenotype and less anthocyanin than the non-inoculated plants Figure 27B). Consistent with leaf phenotype, the total anthocyanin content and chlorophyll content in POT1-inoculated plants were half that of the Al-treated plants and twice that of the Al-treated plants, respectively. Figure 27 F, G). Similar to Arabidopsis thaliana, compared with plants treated with Al, Chinese cabbage plants treated with Al+POT1 showed better growth, such as higher aboveground fresh weight and lower aboveground Al content (F, G). Figure 28 BD).

[0174] Comparison of other Penicillium species with POT1

[0175] To compare the ability of POT1 with other Penicillium strains to alleviate Al stress, Arabidopsis thaliana was grown under high Al conditions in the presence / absence of the following 5 Penicillium strains: *Penicillium baicalensis*, *Penicillium chrysogenum*, *Penicillium micropurpurum*, *Penicillium succinate*, and POT1. Figure 3 A). The results showed that, compared with the uninoculated control plants, the plants inoculated with the fungal strain had longer primary roots, higher root fresh weight, and higher aboveground fresh weight. Figure 29 BD). Interestingly, root fresh weight and aboveground fresh weight were not only the highest in plants inoculated with POT1, but they were also significantly higher than those inoculated with other Penicillium strains. Figure 29 C, D). When Arabidopsis thaliana was grown under high Al conditions in soils uninoculated or inoculated with one of these five Penicillium strains, we observed that plants inoculated with POT1 had the lowest anthocyanin accumulation and the highest chlorophyll content compared to plants inoculated with the other four Penicillium strains. Figure 30 ).

[0176] Summarize

[0177] Increasing phosphate use efficiency

[0178] This invention relates to a newly isolated fungal strain, *Penicillium olsenii* TLL1 (POT1), which has a high phosphate-solubilizing capacity and promotes root elongation in grafted plants. Figure 1 Even with the complete omission of phosphate source in the growth medium, primary root length is longer when POT1 is inoculated. Figure 12 B).

[0179] Due to phosphate deficiency, callose (β-(1,3)-D-glucan) is deposited in the root apical meristem, which hinders intercellular communication necessary for root elongation. Furthermore, nitric oxide (NO) accumulation is an inhibitory factor for primary root elongation. POT1 was confirmed to inhibit callose and NO accumulation in root cells. Figure 2 Despite growth under low phosphate conditions, Arabidopsis roots still elongated normally because callose and NO accumulation was inhibited with root colonization at POT1. Figure 1 H, Figure 12 A).

[0180] Furthermore, iron accumulation at the root tip is the cause of the inhibition of primary root elongation in Arabidopsis thaliana under phosphate limiting conditions. Siderophores are iron-chelating agents produced by bacteria and fungi to scavenge iron from the environment. The evidence presented in this work shows that POT1 is able to secrete siderophores ( Figure 24 ), which indicates that the siderophores secreted by POT1 scavenge iron and reduce iron accumulation in the roots of plants grown under phosphate deficiency.

[0181] Root-associated endophytic fungi are the main contributors to the solubilization of phosphorus in the rhizosphere of Arabidopsis thaliana plants. Phosphorus-solubilizing fungi (PSF) proliferate in the rhizosphere and establish a symbiotic relationship with the plant’s root system. Active species of PSF involved in the phosphate cycle are endophytic fungi such as Penicillium, Aspergillus, Trichoderma, Piriformospora, and Curvularia. Phosphorus-solubilizing fungi can be widely applied as a potential solution to improve the efficiency of phosphate uptake and utilization.

[0182] The insolubility of phosphate fixation in the soil depends on soil factors such as pH, organic matter content, the presence of exchangeable cations such as Fe, Al, Ca, Mg, and the clay content in the soil. At acidic pH (lower than 5.5), iron and aluminum are involved in the phosphate precipitation reaction, while at alkaline pH (pH 6-8), calcium ions start to act, and at pH 7.5, magnesium ions dominate. The lowest fixation of P occurs between pH 6 and 7. Phosphate added to the soil as an agricultural fertilizer reacts with cations such as Al, Ca, and Fe and is fixed in the soil, which is not accessible to plant roots (Penn and Camberato, 2019). The phosphate solubilizing activity of POT1 was confirmed in insoluble phosphate complexes such as tricalcium phosphate, aluminum phosphate, iron phosphate, and hydroxyapatite ( Figure 3 B). POT1 showed high solubilization capacity for tricalcium phosphate in media containing four different phosphate sources.

[0183] It was confirmed here that the growth conditions of Arabidopsis, Brassica chinensis, and rice plants were improved due to the increase in soluble phosphate sources in vermiculite and local Singaporean soil by POT1 ( Figure 4 A, Figure 7 , Figure 8 , Figure 18 , Figure 19 ). Furthermore, comparing the effects of five Penicillium strains in plants showed that POT1 was significantly effective in root growth promotion and phosphate solubilizing activity in phosphate-deficient media and soils containing insoluble phosphate ( Figure 9 , Figure 10). Therefore, it is suggested that POT1 can be used as a potent phosphorus-solubilizing microorganism for the growth of monocotyledonous plants and dicotyledonous crops in fields containing insoluble phosphate and not only applied to upland soils but also to rice soils, showing better activity than other Penicillium strains.

[0184] The application of fungal species as biofertilizers to cultivated land to improve soil quality is becoming an eco-friendly approach to modern agriculture. There are several ways to apply PSF inoculants, such as seed coating, drenching into soil, application on the surface of the plant or root dipping, etc. (Kumar et al., 2018).

[0185] Biofertilizers have been commercialized in the form of dry biomass (dust or granules) and suspensions (water-based and emulsions) (Odoh et al., 2020). Among Penicillium, Penicillium bilaiae, P. italicum, P. albidum, P. Frequentans, P. simplicissimum, P. rubrum, P. expansum, P. oxalicum, and P. citrinum have been commercialized for use as biofertilizers for mobilizing P, Mn, Zn, Fe, Co, Cu, and Mo, and for tolerance to biotic and abiotic stresses (Odoh et al., 2020). Several commercialized biofertilizers formulated with Penicillium (P. bilaiae) can increase P uptake and improve the yield of oilseed rape (Brassica napus) (Chang and Yang, 2009). Other Penicillium species, such as Penicillium radicum and P. italicum, are also used to improve the yield of wheat and soybean (Sneha et al., 2018). P. bilaiae is widely used as a biofertilizer, and several studies conducted in growth chambers and in the field have shown that it increases the biomass, phosphate uptake, and grain yield of wheat (Triticum aestivum L.) (Kucey, 1988), oilseed rape (Brassica napus L.) (Kucey and Leggett, 1989), beans, peas (Vessey and Heisinger, 2001), and lentils (Wakelin et al., 2007).

[0186] Optimal use of chemical fertilizers is necessary to maintain ecosystem functions and develop sustainable agriculture. Therefore, the application, research and method development of biofertilizers need to emphasize the use of effective, stable, non-toxic microbial consortia strains for promoting plant growth. The present invention provides the combined effect of dissolving the insoluble phosphate that plant roots cannot absorb and removing the inhibitory factor of primary root elongation under phosphorus limiting conditions.

[0187] Reducing aluminum stress

[0188] The plant growth promoting potential of POT1 was tested in assays of Arabidopsis seedlings grown on agar plates containing 100 mM Al. In the presence of Al, POT1 was able to significantly promote plant growth. Increases in primary root length, root fresh weight, shoot fresh weight and leaf area were consistent with inoculation of POT1. Notably, Arabidopsis seedlings inoculated with POT1 had better primary root growth, root fresh weight and shoot fresh weight than control plants. The improved growth of plants with added POT1 can be due to the phosphorus solubilizing properties of POT1 (see Example 1) and increased nutrient uptake, as it has been documented that nutrients are transported by hyphae from distant locations beyond the root depletion zone (George, 2000). Similar to plants grown on agar, POT1 provided Al resistance to Arabidopsis and Brassica chinensis grown in soil under Al stress.

[0189] It has been shown that Arabidopsis plants accumulate high levels of Al, Fe, NO and callose in root tips when subjected to Al stress (Zhang et al., 2019). Our microscopy analysis showed that Al+POT1 treatment significantly reduced the accumulation of Al, NO and callose in Arabidopsis roots compared to plants treated with Al only. This result indicates that Arabidopsis plants treated with Al+POT1 do not experience as much Al toxicity as plants treated with Al only, which can be due to POT1-dependent Al detoxification.

[0190] The results show that the Al-tolerant fungus POT1 isolated from the indoor environment shows plant growth promoting traits in the presence of Al. The results suggest that mixing POT1 with organic fertilizers can be a possible strategy to alleviate Al toxicity and promote plant biomass production.

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Claims

1. An isolated fungal strain of the Penicillium olsonii species having the accession number NRRL 68252, deposited on January 18, 2023, at the Agricultural Research Service Culture Collection (NRRL).

2. A plant growth promoting composition comprising the isolated fungal strain of claim 1, mycelium, hyphae, spores, and / or cell-free culture filtrate thereof.

3. The plant growth promoting composition of claim 2, wherein, The composition comprises the culture filtrate or the mycelium of the isolated fungal strain of claim 1.

4. The plant growth promotion composition according to claim 2 or 3, wherein, The composition (a) increases the availability of phosphorus for uptake by plants in a medium for plant growth, (b) reduces aluminum (Al) stress / toxicity in plants grown in a medium comprising aluminum, and / or (c) inhibits negative components of root growth, such as accumulation of iron, nitric oxide, and / or callose in roots.

5. The plant growth promotion composition according to any one of claims 2 to 4, wherein, The composition comprises at least 0.1 g of mycelium.

6. The plant growth promotion composition of claim 5, wherein, The composition comprises at least 0.5 g of mycelium.

7. The plant growth promotion composition according to any one of claims 2 to 6, wherein, The composition comprises at least one phosphorus source.

8. The plant growth promoting composition of claim 7, wherein, The at least one phosphorus source is selected from the group consisting of calcium phosphate, tricalcium phosphate, iron phosphate, aluminum phosphate, and hydroxyapatite.

9. The plant growth promotion composition according to any one of claims 4 to 8, wherein, The medium is an agar medium, a liquid medium, soil, or vermiculite.

10. The plant growth promotion composition according to any one of claims 4 to 9, wherein, The medium comprises at least one of a low phosphorus content, an insoluble phosphate salt, or aluminum.

11. The plant growth promoting composition of any one of claims 2 to 10 for use as a fertilizer.

12. Use of the growth promoting composition of any one of claims 2 to 10 as a fertilizer.

13. Use according to claim 12, wherein, The fertilizer is formulated as dry biomass, preferably dust or granules, or a suspension, preferably water-based or emulsion.

14. A method of promoting plant growth, the method comprising applying an effective amount of the growth promoting composition according to any one of claims 2 to 10 to a medium for plant growth, wherein, Application of the composition promotes plant growth compared to plants grown in untreated medium.

15. The method of claim 14, wherein, The composition (a) increases the availability of phosphorus for uptake by plants in a medium for plant growth, (b) reduces aluminum (Al) stress / toxicity in plants grown in a medium comprising aluminum, and / or (c) inhibits negative components of root growth, such as accumulation of iron, nitric oxide, and / or callose in roots.

16. The method of claim 14, wherein, The medium comprises at least one condition selected from the group consisting of a low phosphorus content, an insoluble phosphate salt, and aluminum.

17. The method of claim 14, wherein, The medium is an agar medium, a liquid medium, soil, or vermiculite.

18. The method of claim 17, wherein, The soil is a rice soil or an upland soil.

19. The method of any one of claims 15 to 18, wherein, The plant is a Brassica species, an Arabidopsis species, or an Oryza species.

20. The method of any one of claims 15 to 19, wherein, The plant is Chinese cabbage, wild-type Arabidopsis thaliana (Col-0), or Oryza sativa var. indica.

21. A plant seed coated with the isolated Penicillium olsonii strain of claim 1 or the composition of any one of claims 2 to 10.

22. The plant seed of claim 19, further coated with a carrier compatible with soil.

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