Entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae play roles of maize (Zea mays) growth promoter

Beauveria bassiana and Metarhizium anisopliae are two of the most important and widely used entomopathogenic fungi (EPFs) to control insect pests. Recent studies have revealed their function in promoting plant growth after artificial inoculation. To better assess fungal colonization and growth-promoting effects of B. bassiana and M. anisopliae on crops, maize Zea mays seedlings were treated separately with 13 B. bassiana and 73 M. anisopliae as rhizosphere fungi in a hydroponic cultural system. Plant growth indexes, including plant height, root length, fresh weight, etc., were traced recorded for 35 days to prove the growth promoting efficiency of the EPFs inoculation. Fungal recovery rate (FRR) verified that both B. bassiana and M. anisopliae could endophytically colonize in maize tissues. The recovery rates of B. bassiana in stems and leaves were 100% on the 7th day, but dropped to 11.1% in the stems and 22.2% in the leaves on the 28th day. Meanwhile, B. bassiana was not detected in the roots until the 28th day, reaching a recovery rate of 33.3%. M. anisopliae strains were isolated from the plant roots, stems and leaves throughout the tracing period with high recovery rates. The systematical colonization of B. bassiana and M. anisopliae in different tissues were further corroborated by PCR amplification of fungus-specified DNA band, which showed a higher detection sensitivity of 100% positive reaction. Fungal density comparing to the initial value in the hydroponic solution, dropped to be well below 1% on the 21st day. Thus, the two selected entomopathogenic fungal strains successfully established endophytic colonization rather than rhizospheric colonization in maize, and significantly promoted its growth in a hydroponic cultural system. Entomopathogenic fungi have great application potential in eco-agricultural fields including biopesticides and biofertilizers.

www.nature.com/scientificreports/ and promoted crop growth, as evidenced by stem height, root length, fresh root and fresh stem weight [17][18][19] . Soil inoculation and foliar spraying of B. bassiana are also the most used application method which considerably enhanced the growth of corn seedlings 20 . The purpose of this study was to evaluate growth-promoting effects and colonization behavior of B. bassiana and M. anisopliae on maize (Zea mays) seedlings and the influence on the plant growth under a hydroponic cultural system.

Effects of B. bassiana and M. anisopliae on the growth of maize seedlings. Both B. bassiana and
M. anisopliae treatment led to an apparently positive effects on maize growth as observed during the 35-day period. As shown in Fig. 1, the growth promotion effect of the fungi on different maize organs is dependent on the growing stages.
Fungal endophytic, temporal and spatial distribution in maize tissues. Isolation and identification of endophytic B. bassiana and M. anisopliae from different tissues of maize. Fungal colonies began to appear 4 days after the cultivation. The spore morphology, mycelial characteristics and colony characteristics of the isolated strains were observed under light microscope. The fungal conidiogenous cell of B. bassiana formed a zigzag curve, and the conidia were spherical, transparent and smooth, consistent with the morphology of the pure strain B. bassiana Bb-13 21 (Fig. 3a). The fungal colonies of M. anisopliae were white in the beginning and turned olive green after sporulation. The hyphae were transparent, separated and branched. Bottle-shaped conidiogenous cells formed a long string of conidia in basal continuity (Fig. 3b). The conidia were elliptical, consistent with the morphology of pure strain Ma73. In general, B. bassiana ( Fig. 3A-C) and M. anisopliae (Fig. 3D-F) were isolated from maize tissues by culturing tissue samples on the fungal selective medium, verifying their endophytic colonization in maize root, stem and leaf.
PCR amplification of special DNA band for B. bassiana(441 bp) and M. anisopliae(150 bp) from different tissues of fungal treated maize seedlings was also used to trace endophytic colonization of B. bassiana (Fig. 4a)       Table 5 showed that the colonization rates of B. bassiana in tissues of all maize organs were 100% in each sample time (7-35 days). The same results were observed in leaf tissues for M. anisopliae, but the fungus was not constantly detected at 100% in maize stems and leaves.

Discussion
When compared with traditional soil cultural systems, hydroponic culture systems are popular for industrial production of vegetables, but not so often for ecological study between microbes and plants. In this study, a hydroponic cultural system instead of a soil cultural system was used to assess fungal colonization and    22 . As monitored by culturing maize tissues on the fungal selective medium, FRRs showed a dependence on the type of plant tissues. Certain endophytic fungi exhibit a degree of tissue specificity as they adapt to existing conditions in each organ [23][24][25][26] . Guo et al. 27 reported that the tissue specificity of endophytic fungi results from their adaptation to the unique conditions of the plant organs.
Inoculation method is crucial to fungal colonization pattern 28 . Parsa et al. 29 found that B. bassiana could colonize endogenously when the plants were treated by spraying or irrigating, while the roots could only be colonized only by irrigation. In sorghum, Tefera and Vidal reported higher rates of B. bassiana colonization in stems by leaf inoculation, and higher rates in roots and stems by seed inoculation. In the current work, root inoculation of both fungi was conducted by adding conidia suspensions directly into the hydroponic cultural system. This method might enhance the fungal transmission efficiency since the flowing water can help the fungal conidia to move toward maize crop roots. Beside inoculation method, other factors such as soil microbes, temperature, relative humidity, growth medium, plant age and species, inoculum density and fungal species may be involved in a successful colonization of fungi in different plant tissues 28 .
Furtherly, PCR amplification of fungus-specified DNA band provides a new and sensitive method for fungal endophytes detection. For example, the FRR of B. bassiana was tested to be low by culturing plant tissues on fungal selective medium, but was tested to be 100% by PCR. Low endophytic fungal population density in plant tissues or biological inhibition from the plant tissues may contribute the failure of fungal growth on selective medium. The PCR amplification method could be reliably used in endophytic fungi study.
Previous studies revealed that some endophytic entomopathogens can promote plants growth as biofertilizers. Jaber et al. 16 reported higher stem height, root length, fresh root weight, and stem weight than uninoculated plants 14 days after inoculating wheat seeds with B. bassiana. Russo et al. 30 reported increased plant height, leaf number, plant height and the number of nodes on the first cob after spraying B. bassiana on maize leaves.
In our study, the two selected entomopathogenic fungi, B. bassiana and M. anisopliae, also significantly promoted the growth of maize in a plant hydroponic cultural system, and established a systematic colonization in different tissues in maize seedlings tissues for an expected long-term growth promotion.
In contrast, Moloinyane et al. 31 did not find any significant differences in plant height, root and leaf number, fresh and dry weight between B. bassiana treated and untreated grapes even 4 weeks after soil drenching treatment. This is not surprising since the endophytic abilities of a particular fungal strain may be strongly associated with host plant species, plant species varieties 22 , nutritional conditions and environmental influences. Tall and Melying 32 studied the effects of B. bassiana (GHA) seed treatment on maize growth. They found that B. bassiana can only be used as a growth promoter of maize only when nutrients were abundant, while no growth promotion effect was noticed in the absence of nutrients. So, mechanisms of plant responses to the fungal endophytism are far from clear and need further examination.
We studied entomopathogenic fungi, B. bassiana and M. anisopliae played their roles as maize growth promoters. However, it is unclear whether, rhizospheric or endophytic is the dominant mechanism? We tracked the population dynamics of B. bassiana and M. anisopliae in plant hydroponic solution and in plant tissues, hoping to shed light on the mechanism. In terms of CFUs, the populations of B. bassiana and M. anisopliae in the hydroponic solution rapidly decreased. After 1 week, a residual percentage of less than 10% of M. anisopliae and 1% of B. bassiana was detected, while both fungi were hardly detected on the 28th day in the maize culturing hydroponic solution. In a control study, both fungal conidia could maintain high viability after 1 week in the hydroponic culture system. Thus, the fungal endophytes, influenced by the conidia adhesion, host recognition and endogenous pathway [33][34][35][36][37] , was the main reason for the sharp decrease of fungal populations in the hydroponic culture system. Furthermore, the growth promotion function of the fungi was mainly due to the endophytic function rather than the rhizospheric function.
Normally, biological functions are density-dependent. The relationship between plant growth promotion and population density of endophytic fungi cannot be clearly clarified until the population of endophytic fungi in plant tissues could be quantitatively detected. The plant growth promotion mechanism still needs further exploration in the entomopathogenic fungi-plants interaction system. The entomopathogenic fungi exhibit great potential not only on both bio-control of pests but also on plant growth promotion, bringing us a new sight on ecological relationship among plants, insect pests, and entomopathogenic fungi. www.nature.com/scientificreports/ The conidia suspension of B. bassiana and M. anisopliae strain were inoculated on PDA and SDAY medium respectively. After 14-18 days of cultivation, conidia were harvested by scraping conidia from the agar surface with a sterile inoculation shovel, and certain amount of conidia was put in sterilized 0.05% Tween-80 (v/v) and which was well mixed to make fungal conidia suspensions 38,39 . Conidial concentrations were determined using a hemacytometer and the final suspension was adjusted to 1 × 10 7 conidia mL −1 .

Methods
Plants. Maize (Z. mays, Jingnuo No.1) seeds were purchased from Shouguang Yino Agricultural Science and Technology Co., LTD. The maize (100 seeds for one treatment) were seeded in breeding discs, each one with 32 holes (5.4 × 6 × 5 cm) containing sterilized culture substrates (Klasmann peat, German) (about 30 g of sterile soil is placed in per hole). One seed was placed per hole one seed. All maize seedlings are cultivated in a greenhouse for 14 days with automatically controlled environmental factors (25 ± 1 °C, 70 ± 5% RH and 16L: 8D regime).
Fungal inoculation of maize seedlings. Ninety maize seedlings with even and well-good growth were randomly sampled for each treatment. The culture substrates on roots of each maize seedling were gently washed out with distilled water, avoiding any damage to the roots. The treated maize seedlings with even growth condition including aboveground and underground parts were transplanted to a maize hydroponic culturing system.
The maize hydroponic culturing system was constructed with an opaque plastic box (Working box specification: length × width × height of = 67 × 42 × 17.5 cm 3 ) covered by an opaque plastic plate with 40 holes, in which 45L 1/2 half Hoagland nutrient solution (1/2) was added as hydroponic nutrient solution. A maize seedling was carefully transplanted in a culturing hole by fixing the plant stem with a sponge block 40 . Oxygen supply for the maize hydroponic culturing system was supported by a small ventilation pump (Risheng Group Co. LTD., CHN).
After two-day domestication for maize seedlings, 100 ml of conidia suspension with 1 × 10 7 conidia mL −1 was added into each hydroponic solution. Eighty plants were used as the control, in which 100 ml of sterile 0.05% Tween-80 (v/ v) was added.

Plant growth measurements.
Maize seedlings were weekly sampled at random for each fungal treatment and the control. All plant growth indexes including plant height, root length, leaf length, leaf width, number of leaves, and fresh weight (the above-ground, underground and the total) were carefully measured 20 . The weight of above and below ground parts was separately weighed in an analytical balance (Yue Ping Scientific Instrument Co., LTD., CHN). All measurements were conducted by eight randomly sampled maize seedlings with two separate batches.

Fungal population dynamic in hydroponic solution. Population density of B. bassiana and M.
anisopliae in hydroponic solution were periodically monitored by the 7th day. One milliliter hydroponic solution was randomly sampled 5 times at a depth of 2 cm with a micropipette. The sampled hydroponic solutions were gradient diluted to 10, 100, 1000, 10,000 times with 0.05% Tween 80. After that, 100 uL of hydroponic solutions at 5 concentrations was sampled and evenly spread on a petri dish for fungal growth. Five repeats of BSM (Beauveria selective medium, quarter strength PDA containing 350 mgL −1 streptomycin sulfate, 50 mgL −1 tetracycline hydrochloride and 125 mgL −1 cycloheximide) petri dishes were used to assess the fungal population by terms of CFUs. The petri dishes were incubated at (25 ± 1) °C for fungal growth. The fungal colonies on the petri dishes were counted and identified. The optimal diluted concentration of the sampled hydroponic solution was the CFU number between 10 and 100 CFUs/plate 41 . The CFUs in the optimal range was used to value population dynamic in the hydroponic solutions.
Determination of fungal colonization in maize tissues. Recovery of entomopathogenic fungi from maize tissues. The fungus was re-isolated by culturing tissues of the roots, stems and leaves of maize seedlings, inoculated with B. bassiana or M. anisopliae as rhizospheric fungus, on fungal selective medium to confirm fungal endophytic. Firstly, tissues of roots, stems and leaves were cut into pieces with 0.5 cm of length (roots and stems) or 0.5 cm 2 (leaves), and the small pieces of tissues were sterilized with 1% sodium hypochlorite for 5 min, then soaked in 75% ethanol for 1min 42 . Washed three times in sterilized distilled water, the tissue samples were inoculated on BSM medium and incubated at (25 ± 1) °C in the dark. Efficiency of the sterilization procedures of plant tissues was evaluated by inoculating 100 ul of the last rinse water on PDA medium to check any bacteria or fungi growth.
The BSM petri dishes were daily checked to find whether fungal colony appeared or not. Each growing fungal colony on BSM medium was inoculated on PDA or SDAY medium to identify the fungal species. DNA extraction of maize tissues. All maize tissues(100 mg) for DNA extraction were sampled and sterilized as the above procedure and then cut into small pieces with sterilized surgical scissors. Each tissue sample was transferred into a 1.5 ml centrifuge tube with steel balls added 39 . The tube was put into liquid nitrogen for 2 min frozen, and then the tissue sample was quickly ground for 150 s in an automatic grinding machine (Jingxin Technology Co., LTD, Shanghai). DNA was extracted from the sample tissue followed the steps of fungal genomic DNA extraction kit (Vokai Biotechnology Co., LTD., Beijing). The extracted DNA quality was exam-  -TTC CGA ACC CGG TTA AGA GAC-3' , R5'-TTC CGA ACC CAT CAT CCT GC-3') 44 or M. anisopliae (5'GAC TCT CTT AAG GTA GCC AAA TGC C3' , 5' AAA CTC CCC ACC TGA CAA TG-3') 45  Statistical analysis. All experimental data were analyzed using IBM SPSS Statistic (Version 20.0) for univariate ANOVA analysis, and Turkey HSD was used to test the significance of differences among treatments (P ≤ 0.05).

Plant material collection and use permission.
No permission is required for plant material as it was purchased from certified dealer of local area. The use of plants or plant materials in the present study complies with international, national and/or institutional guidelines.
Ethics approval and consent to participate. The study has been conducted without violating any ethical codes of conduct.

Conclusions
In summary, two entomopathogenic fungi, B. bassiana and M. anisopliae, played positive roles in promoting the growth of maize seedlings after hydroponic rhizosphere inoculation. The two fungi could establish systematic colonization in tissues of all maize organs through maize roots, and established within 1 week. Fungal population dynamics in hydroponic solution and fungal colonization in maize tissues suggested that the fungal endophytic function other than rhizospheric function contributes more to the observed plant growth promotion. Fungal endophytic behavior was found to be somewhat fungal species specific. The technique of PCR amplification of special DNA bands of the fungi is a more sensitive method than the fungal colony detection method by culturing plant tissues on fungal selective medium. The technique could be used to trace fungal colonization and spatial distribution in plant tissues with higher accuracy. Further study is still needed to elucidate the mechanisms of the responses of the plant and plant pests to the fungal endophytism (Supplementary Information).

Data availability
The datasets generated during the current study are available from the corresponding author on reasonable request.

Funding
This research was funded by the Key Project of Natural Science Research of Anhui Universities (KJ2020ZD08).