Phosphate solubilizing fungi isolated and characterized from Teff rhizosphere soil collected from North Showa zone , Ethiopia

Phosphorus is one of the major bio elements limiting agricultural production. About 95 to 99% phosphorus in agricultural soil is unavailable form for plant growth. Phosphate solubilizing microorganisms can increase soil phosphate availability. This study was aimed to identify and evaluate phosphate solubilizing fungi from Teff rhizosphere soil. Fungi were identified using lactophenol cotton blue staining confirmation and Biolog Microstation identification system. Fungi isolates were screened and transferred to biolog universal yeast agar media. Pure yeast cells and filamentous fungi were suspended in sterile water and filamentous fungi (FF) inoculum fluid at 49±2 and 75±2 turbidity measured by biolog turbidimeter, respectively. 100 μL transferred from each suspension into 96 wells of the biolog yeast microplate and filamentous fungi microplate tagged with different carbon source and incubated at 26°C for 24 to 72 h and read by micro station at a single wavelength of 590 nm, results were recorded and processed for identification by micro log3 software ver. 4.20.05. Biolog microstation read 24 fungi species. Filamentous fungi ≤0.5 similarity index (62.5%), yeast 0.5 similarity index (25%), yeast ≤0.5 similarity index (12.5%). The identified fungi were tested for phosphate solubilization by the Pikovskaya’s agar (PVK) selective media. Seven species were positive in phosphate solubilizing ability: Trichosporon beigelii B, Rhodotrula aurantiaca A, Cryptococcus luteolus, Zygoascus hellenicus, Penicillium purpurogenum var. rubrisclerotium, Neosartorya fisheri var. fischeri, and Candida montana. At 15 days incubation, T. beigelii B and R. aurantiaca A was able to solubilize phosphate with solubilizing index of 5.3 and 2.6, respectively. T. beigelii B, were superior in phosphate solubilization. Therefore, these species can be candidated and exploited after further evaluation as biofertilizers for agriculture productivity.


INTRODUCTION
Phosphorus (P) is the second essential macronutrient for plant growth and development.It accounts 0.2% of plant dry weight, limits the growth of plants and crop yield (Sharma et al., 2013).Phosphorus contributes remarkably to photosynthesis, energy and sugar production, nucleic acid synthesis, and promotes N 2 fixation in legumes (Saber et al., 2005).
The mineral nutrition of plants mainly depends on soil P content that can be assimilated as a soluble phosphate (Ehteshami, 2011).Phosphorous increases the strength of cereal straw, promotes flower formation and fruit production, stimulates root development and also essential for seed formation (Sharma et al., 2011).It also plays a role in stalk and stem strength, maturity and production crop quality and resistance to plant diseases (Richardson, 2007).Mobility of phosphate ions in the soil is very low due to their high retention in soil.Stevenson (1986) and Holford (1997) reported that the recovery rate of P fertilizer by plants is only about 10 to 30%.The remaining 70 to 90% is accumulated in soil or in the form of immobile that is bound by Al or Fe in acid soils, or Ca and Mg in alkaline soils (Prochnow et al., 2006;Yang et al., 2010).Phosphorus is highly insoluble and unavailable to plants.It must be converted into soluble form.Phosphate solubilizing microorganisms can play an important role in dissolving both fertilizer phosphorus and bound phosphorus in the soil that is environmentally friendly and sustainable (Khan et al., 2007).Several groups of microorganism including fungi, bacteria and actinomycetes are known as efficient fixed P solubilizers (Sundara et al., 2002).Fungi are the important components of soil microbes typically constituting more of the soil biomass than bacteria, depending on soil depth and nutrient conditions.Fungi have been reported to have greater ability to solubilize insoluble phosphate than bacteria (Nahas, 1996).A wide range of soil fungi are reported to solubilize insoluble phosphorous such as Aspergillus niger and Penicillium species, which are the most common fungi capable of phosphate solubilization (Whitelaw et al., 1999).Exploration of phosphate solubilizing microorganisms has been conducted by many researchers from soils (Chen et al., 2006;Widawati et al., 2008;Gupta et al., 2012), mangrove (Vazquez et al., 2000;Holguin et al., 2001), and rhizosphere (Chung et al., 2005;Poonguzhali et al., 2008;Oliveira et al., 2009).From such explorations various types of phosphate solubilizing microorganisms have been successfully identified.In last few decades, a large array of rhizosphere bacteria and fungi including species of Penicillium, Azotobacter chroococcum, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Arthrobacter ilicis, Escherichia coli, Pantoea agglomerans, Pseudomonas putida, Pseudomonas aeruginosa, Enterobacter aerogenes, Microbacterium laevaniformans, and Micrococcus luteus have been identified as P-fertilizers (Kumar et al., 2014).The principal mechanism for many soil fungi and bacteria can solubilize inorganic phosphate into soluble form through the process of acidification, chelation, exchange reactions and production of organic acids (Han, 2006).Acid phosphatases play a major role in the mineralization of organic phosphorus in soil phosphate solubilization effect is mainly through the reaction between organic acids excreted from organic matters with phosphate binders such as Al, Fe, and Ca, or Mg to form stable organic chelates to free the bound phosphate ion (Arcand and Schneider, 2006;Gupta et al., 2012).Phosphorus deficiency is the most important problem of Ethiopian soil and more than 70 to 75% of highland soils are characterized by phosphorus deficiency (Beyene, 1982).The deficiency is very severe in the acidic soils of the southern, southwestern and western regions.Areas Al 3+ and Fe 3+ high are totally incriminated with phosphorus fixation (Sertsu and Ali, 1983).The fixed forms of P in acidic soils are aluminum and iron phosphates, while in alkaline soils they are calcium phosphates (Rfaki et al., 2014).Around 70% of Ethiopian vertisol have available phosphorus below 5 ppm, which is very low for supporting good plant growth and fixation in vertisols is related more to calcium, which is the predominant cation in all profiles than Al 3+ and Fe (Mamo et al., 1988).Vertisols are dark, montmorillonite-rich clay soils with characteristic shrinking and swelling properties.They have high clay content (>30% to at least 50 cm depth from the surface) and when dry they show cracks of at least 1 cm wide and 50 cm deep.They have high calcium and magnesium contents (FAO, 2000).Teff [Eragrostistef (Zucc.)Trotter] is the major indigenous cereal crop of Ethiopia, where it was originated and diversified.It is a highly demanded and a staple food grain for majority of the Ethiopian people.In a country of over 80 million people, teff accounts for about 15% of all calories consumed in Ethiopia (Bekabil et al., 2011).The teff grain is ground to flour which is mainly used for making popular pancake-like local bread called injera and sometimes for making porridge.The grain is also used to make local alcoholic drinks, called tela and katikala.Tef straw, besides being the most appreciated feed for cattle (Ketema, 1997).Teff is the only cultivated of all 300 Eragrostis species.Its agro ecological adaptability has resulted in its cultivation as an important crop in 10 of 18 agro ecological zones of the country.It can be grown in altitudes ranging from near sea level to 3000 ms, but the best performance occurs between 1100 and 2950 masl (Hailu and Seyfu, 2000).Annual rainfall of 750 to 850 mm, growing season rainfall of 450 to 550 mm and a temperature range of 10 to 27°C.A very good result can also be obtained at an altitude range of 1700 to 2200 m and growing-season rainfall of 300 mm (Seyfu, 1993).The crop performs well in both water logged vertisol in the highlands as well as water-stressed areas in the semi-arid regions throughout the country and consequently it is preferred over other grain crops such as maize or barley (Zeleke, 2010).Teff production and productivity have been far below the potential (Demeke, 2013).Currently, the average national productivity is estimated to be less than 0.5 ton per ha.This is very low compared to other cereals such as wheat and sorghum grown in the region.Lower grain yield is mainly attributed to low soil fertility, especially nitrogen (N) and phosphorus (P) deficiencies and weed control practices (Seifu, 1993).Declining soil fertility as a result of continuous cropping without replenishing soil nutrients, continues application of phosphate fertilizer and soil erosions is the major factors that reducing production and productivity of the crop in Ethiopia.Higher grain yield of teff was recorded by applying inorganic fertilizers (Abate, 1993).However, chemical fertilizers are neither easily available nor affordable for the majority of poor Ethiopian farmers and not environmentally friendly (FAO, 1987).Such economic considerations necessitate for an alternative less expensive and environmentally friendly agricultural technologies to improve yield and quality of grain.Screening and characterization of phosphate solubilizing microorganisms are important for proper utilization of their beneficial effects to increase crop production and sustain agricultural productivity of the country without contaminating environments.In Ethiopia, only few studies on teff root-associated microorganisms have been undertaken.The effect of phosphate solubilizing some fungus on growth and yield of teff was studied by Asfaw (1993).Inoculation of teff by vascular arbuscular mycorrhazal (VAM) and plant growth promoting rhizobacteria (PGPR) give good result on teff productivity.So previous research works tell us using biofertilizer is better indicative to improve teff productivity to a significant level.However, there are some trials on rhizobacteria and vascular arbuscular mycorrhazal using as biofertilizer, phosphate solubilizing fungi were not studied well.This study was aimed to isolate, identify and evaluating of phosphate solubilizing fungi from teff rhizosphere soil collected from North Showa farm land and selecting superior solubilizing fungi that will be candidated for bio fertilizer after further evaluation for agricultural productivity.

Study area
The study was conducted in North Showa zone in five selected districts, particularly in Kewot, Tarmaber, Efratana gidim, and Siadeberna wayu.North showa zone is one of the 10 zones of Amhara regional state.The elevation ranges from 1100 to 3009 m above sea level.Geographic coordinate latitude: 9°46'8.4"and longitude: 39°40'4.8".The zone is located in approximately average 200 km far from Addis Ababa (Figure 1).

Sample collection
Twenty five (25) teff farmland site were selected based on three teff varieties, two soil types and 200 m difference within 1200 to 2200 m.a.s.l altitude in the study area.Seventy five rhizosphere soil samples were collected through drillings at 5, 10, and 15 cm depth (Figure 2).Approximately, 15 g of soil were taken from each depth of sampling point and a total of 45 g composite soil per sampling

Screening and isolation of fungi from teff rhizosphere soil
One gram of soil from each sample was serially diluted up to 10 -6 mL in distilled water.About 0.1 mL inoculum sample was transferred to yeast extract peptone dextrose agar media (YPDA), rose bengal agar, potato dextrose agar by cotton swab and streaked using nichrom loop.Primary cultures were incubated for 26°C in digital incubator for 48 h.Isolates were subculture twice until pure colony was obtained for morphological identification.A single yeast colony and pure filamentous fungi was streaked to Biolog universal yeast agar (BUY agar plate (60 g/1 L) and incubated for 48h at 26°C for yeast and filamentous fungi micro plate (YT/FFMicroplate) inoculum preparation).The yeast and filamentous fungi were identified according to the Biolog micro station reading and procedure.

Colony morphology identification
The colony morphology of the isolated fungi were examined after grown on yeast extract peptone dextrose agar media and biolog universal yeast agar media at 26°C for 48 h and its colony morphology, form, size, elevation, margin/edge, and colony color were observed using hand lens as well as its percentage frequency were recorded.

Identification of yeast from teff rhizosphere soil
Pure yeast isolates after being grown on yeast extract, potato dextrose agar were transferred to biolog universal growth agar and incubated at 26°C for 48 h.Pure colony of yeast suspensions were prepared in 9 mL sterile distilled water and adjusted to 47±2T using biolog turbidiameter. 100 µL of inoculum was dispensed using digital pipettor to each of 96 wells of yeast micropate (YT) and incubated at 26°C 24 to 72 h.
The YT micro plate is tagged with 96 carbon source.An isolate ability to metabolize each carbon source is measured in the presence or absence of purple hue in the wells.Tetrazolium violet a redox dye forms a purple color when oxidized by cellular respiration of microorganisms.The YT micro plate measures both metabolic reactions as well as turbidity growth to produce identifications.YT micro plate was read by the micro station reader at 24, 48, and 72 h at a single wavelength of 590 nm.The biolog software micro log3 ver.4.20.05compared the results obtained with the test strain to the database and provided identification based on distance value of match and separation score produces similarity index value and probability for species identification (Biolog1993).

Identification of filamentous fungi from teff rhizosphere soil
Filamentous fungi screened and isolated on rose bengal agar and potato dextrose agar were stained by lactophenol cotton blue in order to confirm to which genera the fungi is belonged to then pure filamentus fungi were transferred into biolog universal growth agar media and incubated at 26°C for 48 h.Pure sporulate filamentous fungi suspension were prepared using 15 mL filamentous fungi inoculum fluid and adjusted to 75±2T using biolog turbidiameter. 100 µL of inoculum was dispensed using digital pipettor to each of the 96 wells of filamentous fungi micropate (FF) tagged with different carbon source and incubated at 26°C and 24 to 240 h.After incubation, the FF micro plate measures both metabolic reactions as well as turbidity growth to produce identifications.Filamentous fungi micro plate (FF) was read by the micro station reader at 24, 48, and 72 h at a single wavelength of 590 nm.The biolog software micro log3 ver.4.20.05compared the results obtained with the test strain to the database and provided identification based on distance value of match and separation score produces similarity index value and probability for species identification (Biolog1993).

Identification of phosphate solubilizing microorganisms
Fungal isolate identified by biolog microstation were tested for their phosphate solubilizing ability.Pure fungi colonies were collected using a needle nose and spotted at 4 quadrants on sterile solid Pikovskaya media (2.5 g Ca3(PO4), 0.5 g (NH4)2SO4, 0.2 NaCl, 0.1 g MgSO4.7H2O,0.2 g KCl, 10 g glucose, 0.5 g of yeast extract, 20 g agar, 0.0001 g MnSO4, 0.0001 g FeSO4, and 1000 mL distilled water) (Rao, 1982).Ca3(PO4)2 was used as a source of phosphate.Observations were made until the formation of a clear zone around the colonies of fungi that indicated the occurrence of phosphate dissolution.At 5 days intervals, solubilization index (SI) was measured using the following formula (Premono et al., 1996).Fungi that formed the fastest clear areas with the greatest diameter indicate the most superior phosphate solubilizing fungi.

Statistical analysis
The data analysis involved various descriptive statistics such as means and percentages frequency.STATA ver.13 was used for phosphate solubilization index data analysis.

Percentage frequency of fungal species isolated from teff rhizosphere soil
A total of 450 fungal colonies were grown and counted on different growth media and identified pure colonies having similar morphology were clustered in order to detect the incidence frequencies of the microorganisms encountered.Sixty five percent were filamentous fungi and 35% were non filamentous fungi.From filamentous fungi, Aspergillus species were dominant (33%), Penicillium species (29%), Fusarium species (16%), Trichoderma (13%), and Colletotrichum (9%).The phosphate solubilizer fungi isolates were also identified based on their colony morphology that is pigmentation, shape, size, texture, elevation and margin) (Table 1).

Identification of filamentous fungi species using lacto phenol cotton blue staining (LPCB) and biolog micro station
Representative filamentous fungal isolates from clustered group were stained using lacto phenol cotton blue to confirm to which genera filamentous fungi belonged to kept in +4 o C until processed.2).

Phosphate solubilization test
A total of 24 fungus species were evaluated for their phosphate solubilization efficiency on Pikovskaya's agar selective media.Among all 7 isolates were positive for phosphate solubilization (Table 4).From 1.0 to 3.4 cm clear zone diameter were recorded within 15 days of incubation (Figure 4).T. beigelii B showed superior solubilization index (PSI) of 5.3, followed by R. aurantiaca A which is 2.6, the smaller solubilization index recorded 1.5 by P. purpurogenum var.rubrisclerotium (Table 4 and Figure 4).

Conclusion
Twenty four (24) fungi isolated from teff rhizosphere soil using lactophenol cotton blue staining and biolog microstation identification system where equivalent to molecular techniques and the dominant species were filamentous fungi.Seven fungi species T. beigelii B, R. aurantiaca A, C. luteolus, P. purpurogenum var.rubrisclerotium, Z. hellenicus, N. fisheri var.fischeri, and C. montana were positive for phosphate solubilization efficiency.T. beigelii B was the superior among the isolated fungi in solubilizing index of 5.3 followed by R. aurantiaca A with 2.6 and good candidate after further evaluation on in vitro test, green house and field trials as bio fertilizer.The rise in the cost of chemical fertilizer, the lack of fertilizer industries in developing countries and the growing environmental issue and biodiversity loss using chemical fertilizer are timely important concern using alternative ecofriendly bio fertilizer to increase yield and productivity of teff crop.

RECOMMENDATION
The beneficial effects of plant growth promoting microorganisms (PGPM) have not been exploited well.In the past, some microbial inoculants prepared from Rhizobium for leguminous crops, Azotobacter and Azospirillium for cereal crops and Frankia for tree crops have been used as nitrogen providers in many developed and developing countries.However, enormous interest increase in research in recent years in PGPM such as nitrogen fixer, phosphate solubilizer, and pathogen suppressor.There is no well-organized microbial inoculant industry for bio fertilizer production especially for phosphate solubilizer and there is no link with researcher working on microbial bio fertilizer in Ethiopia, therefore, Agricultural Research Institute, microbiologist, soil scientist agronomist, and stockholders in general must work together in depth on structural and functional diversity of PGPM and selecting superior biofertilizer, biopesticide, biostimulant increase crop yield and prductivity.Further research should be continued with selecting efficient phosphate solubilizer microorganism (PSM) isolates.These may be used for inoculum production and their inoculation effect on the plant growth must be studied in vitro, green house and field trials.

Figure 1 .
Figure 1.Map of study area.

Table 2 .
Biolog micro station filamentous fungi identification result read.

Table 3 .
Biolog micro station yeast identification result read.