Plant growth-promoting fungi from desert soil

: Bio-fertilizers can be expected to reduce the use of chemical fertilizers and pesticides, but they are not yet able to replace their use. Bio-fertilizer production is cheap and does not create pollution in the natural system. Plant growth promoting fungi (PGPF) play important roles in plant nutrition, soil biology and soil chemistry, (PGPF) enhanced photosynthetic rate and other gas exchange-related traits as well as increased water uptake. PGPF are common root-associated and soil-borne fungi from diverse genera, which have the natural ability to stimulate various growth related traits of plants. Plant growth promoting fungi (PGPF) were isolated from various soils of Aswan University Campus, South–western Desert, Aswan, Egypt. The purified isolates were classified as Trichoderma longibrachiatum and Fusarium solani according to phylogenetic analysis and were used as bio fertilizers and to enhance Vigna unguiculata growth under irradiance stress. In current study it was found that these isolates improve Vigna. unguiculata growth under irradiance stress through increasing chlorophyll content compared to control and showing maximum photosynthesis rate ( Pn ) at high Photosynthetic Active Radiation ( PAR ). It was concluded that Trichoderma longibrachiatum and Fusarium solani possessed multiple beneficial effects for Vigna. unguiculata productivity when grown under harsh environmental conditions.

PGPF and PGPR are known to suppress some plant diseases.Trichoderma species are among the most commonly studied as bio-control microbes, also have been shown to induce local and systemic defense responses in agricultural crops.Although, it has also direct effects on plants, increasing or accelerating their growth, resistance to diseases and the tolerance to a biotic stresses, some Trichoderma strains are commonly used as bio-control agents in agriculture due to their abilities in antibiotics and/or hydrolytic enzymes production that directly antagonize plant-pathogenic fungi (Benítez et al., 2004).Some Trichoderma strains also enhance plant growth and development, and thus increase yields.Trichoderma spp are known to have the capability of attacking other fungi.Also they are well known as potential biological control agents and can kill plant pathogens and improve the plant growth (El Komy et al., 2015;Naher et al., 2014;Sundaramoorthy and Balabaskar, 2013;Contreras-Cornejo et al., 2015a, Contreras-Cornejo et al., 2015b;Garnica-Vergara et al., 2016).Besides, Trichoderma spp have been proven its ability to detoxify toxic compounds and fasten degradation of organic materials (Amira et al., 2011;Sharma et al., 2012;Vázquez et al., 2015;Zafra et al., 2015).The success of Trichoderma spp in the soil ecosystem and its role as natural decomposer is due to its ability to hasten the growth, by nutrient uptake and modifing the rhizosphere.It is also able to tolerate unfavourable environment and has powerful destructive capability against plant pathogenic microorganisms ( Benítez et al., 2004).Vigna is well adapted to stress and has excellent nutritional qualities.It is a key dietary staple for the poorest sector of many developing countries and greatly improves an otherwise bland and unbalanced diet (Som and Hazra, 1993).It is one of the most ancient human food sources and has probably been used as a crop plant since Neolithic times.Vigna is the crop of all-round utilization that grown for dry seed, immature seed, immature green pod, green leaves, and even roots.It is one of legumes that is an important source of nutrients and provides high-quality, inexpensive protein to diets based on cereal grains or starchy food (Harman, 2006).

Media Employed to Isolate Fungi
Trichoderma sp.isolation and other important prospective fungi candidates: Tsm, or the Trichoderma Selective Media Bengal agar-based Martin's rose media (MRB), The following ingredients were included in the basal medium (g/l distilled water): 0.1 g chloramphenicol, 0.15 g rose bengal (BDH Chemicals), 0.15 g KCl, 1.0 g N H 4 NO 3 , 3.0 g glucose, 0.22 g MgSO 4 (7H 2 O), and 20 g agar.950 ml of distilled water with these ingredients was autoclaved at 121 °C for 15 minutes.The biocidal components were 0.2 g each of captan and quintozene.According to Elad and Chet (1983), these were combined with 50 ml of sterilized (autoclaved at 121 °C for 15 min) distilled water and put to the autoclaved basal medium.
In order to make potato dextrose agar medium (PDA), 200 grammes of potato infusion, 20 grammes of dextrose, and 20 grammes of agar were combined with 1000 ml of distilled water and autoclaved at 121 degrees celsius for 15 minutes.

Identification of the Isolated Fungi by Morphology
On the basis of their culture properties (colour, texture, and pigmentation), spores, and spore-bearing structure, all the isolated fungal colonies were morphologically identified.The fungi's genus and species were identified using the sources below: 1. Barron, (1968) for the genera of Hyphomycetes.2. Domsch & Gams, (1972) for fungi in general.

Genotypic characterization of isolated fungi
Genotypic characterization was carried out using a small subunit ribosomal RNA gene, partial sequence; internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence (Wolf et al., 2004).

An Infection Test
During the experiment, pots holding 4 kg of sterile sand-clay soil were prepared for planting in the greenhouse (1:2).Healthy V. unguiculata seeds, meantime, were sterilized in 70% ethanol for 2 minutes before being thoroughly cleaned with sterile distilled water and prepared for planting.Each pot was planted with two sterilized seeds, and our experiment was divided into eight groups, each with ten pots.The first group served as the control and received no inoculation; the second group received an inoculation with calcium (egg shells) and phosphate, and the third group received Trichoderma spore suspension.Fusarium spore suspension was used in the fourth group's inoculation, Trichoderma spore suspension was used in the fifth group's inoculation, along with Phosphate, Fusarium spore suspension was used and Calcium in the sixth group's inoculation, and a mix of Trichoderma and Fusarium spore suspension was used in the seventh group's inoculation.The last group was inoculated with mix of Trichoderma, Fusarium spore suspension in addition to Phosphate and Calcium.For 73 days, all of the experiment-specific pots received frequent irrigation to maintain the soil's 9% moisture content.

Analysis of Photosynthesis (Pn), Water Use Efficiency (WUE) and Transpiration Rates (E) in Plants
According to readings from the Research Unit for Study Plants of Arid Lands (RUSPAL)'s meteorological station, photosynthesis (Pn) and transpiration rate (E) were measured for plants grown under irradiance stress at photosynthetic active radiation (PAR) ranges from 0 to 2500 mol m -2 s -1 at the 9 th week prior to plants growing (Ali et al., 2018).The following formula was used to calculate water use efficiency (WUE), as described by Silva et al. (2013) , Ali et al. (2018) and, Radwan andSaleh (2022).
WUE was calculated from the ratio: WUE=Pn/E

Statistical analysis
Statistics were used to analyze each parameter that was measured.MINITAB12 statistical software, developed in the USA (Min-itab Inc., 1998), was used to do a two-way analysis of variance.Data are displayed as mean SE.

16S rDNA Gene Sequence of Isolated Strains
Two fungal strains that were isolated from our study were identified as Trichoderma longibrachiatum T1 and Fusarium solani F2 (Figure 3) with accession numbers: (OQ615796), (OQ615913), respectively, as deposited in the Gene bank (Figure 2a, b).
According to two-way analysis of variance, substantial ratio changes were linked to several phonological phases, with Fv/Fm versus treatment being associated with F =2310.65 and P ≤ 0.0001, respectively, under the PAR ranges (0-2500 molm -2 s -1 ).

The role of PGPF in alleviating irradiance stress:
The survival, toleration, competence, colonization, and expression of desired features in the plant-soil system are all influenced by both biotic and abiotic variables.The ability of plants to respond to abiotic conditions such salt, drought, heavy metal toxicity, severe temperatures, and oxidative stress is strongly influenced by their microbial communities.According to recent studies, the fitness advantages provided by some PGPF help plants adapt to stresses (Khan et al., 2011).Even in less-than-ideal settings, there have been reports of increased plant growth due to the connection of PGPF with plants (Bae et al., 2009).
Trichoderma spp., which are free-living fungi that are widespread in soil and root ecosystems and have been known for many years to create a variety of antibiotic substances as well as parasitize other fungi (Denis and Webster, 1971), are known to produce a wide range of antibiotic substances.According to recent research, they are virulent, opportunistic plant symbionts that cause systemic resistance in plants similar to rhizo-bacteria (Harman et al., 2004).These symbionts are well known for their amazing interactions with host plants and their capacity to provide broad-spectrum resistance to plant diseases (Harman et al. 2004).
Decreases in Fv/Fm are typically noticed when plants are subjected to abiotic and biotic stresses (such drought, heat, salinity, cold, or pathogen infection) in the sun.Because it is such a common occurrence, Fv/Fm measurements offer a quick and easy method of tracking stress.Unfortunately, the causes of reductions in Fv/Fm brought on by stress are sometimes complicated.Increases in non-photochemical quenching processes can lead to a drop in Fm when light-stressed photosynthetic tissues.Such quenching causes drops in Fv/Fm and may not recover over a brief time of dark adaption, or even overnight (Adams and Adams, 2006;2004).
Increases in non-photochemical quenching are frequently accompanied by photo inactivation of the PSII reaction centre under stress, which causes excitation energy to be released as heat rather than through photochemistry (Melis, 1999).According to Aro et al. (1994), photo in activation can result in oxidative damage and the loss of the PSII reaction centre, both of which are linked to an increase in F0 (Bradbury and Baker, 1986;Osmond, 1994).
However, it can frequently be challenging to pinpoint the underlying causes of such declines.Since water loss was accompanied by strong irradiance at all stages, the parameters of chlorophyll fluorescence were primarily affected.In the assimilatory organs of H. muticus, chlorophyll fluorescence parameter values showed a statistically highly significant negative that was related to water deficiency stress (Mihaljevi et al., 2021).