EFFECT OF Trichoderma harzianum AND Aneurinobacillus migulanus APPLICATIONS ON AUXIN, GIBBERELLIC ACID AND ABSCISIC ACID CONTENT OF GLADIOLUS CORMS INFECTED WITH Fusarium oxysporum f. sp. Gladioli

Growing gladiolus corms in soil-less culture fertilized by soluble nutrients raised the question of how both Trichoderma harzianum and Aneurinobacillus migulanus enhance plant growth and provide protection against infection by Fusarium oxysporum f. sp. gladioli. Changes in the concentrations of the plant growth regulators (PGRs) gibberellic acid (GA3), indole acetic acid (IAA) and abscisic acid (ABA) were determined in gladiolus corms following treatment with T. harzianum, A. migulanus and F. oxysporum f.sp. gladioli. The presence of these PGRs in corms was confirmed using liquid chromatography-mass spectrometry (LC-MS/MS) and the compounds quantified using high performance liquid chromatography (HPLC). Concentrations of both GA3 and IAA increased linearly in all corms over the 10 days after treatments; lowest concentrations of both PGRs were consistently found in corms inoculated with F. oxysporum f. sp. gladioli. In contrast, highest concentrations of both GA3 and IAA occurred in corms treated with T. harzianum alone. Highest concentrations of ABA, however, were found in corms inoculated with F. oxysporum f. sp. gladioli, regardless of the presence of T. harzianum or A. migulanus. Treatments with T. harzianum or A. migulanus in the absence of F. oxysporum f.sp. gladioli maintained ABA concentrations at the same levels as control corms. These results indicate that promotion of Gladiolus growth by T. harzianum or A. migulanus may be partly mediated through stimulation of the production of GA3 and IAA in corm tissues. Increases in these PGRs may also be a mediator in the biological control activity shown by T. harzianum or A. migulanus.


INTRODUCTION
Plant growth regulators (PGRs) are organic compounds naturally occurring in plants, which regulate many processes in plant development. Since the discovery of PGRs and their varying effects, it has become evident that these compounds play an important role in plant-pathogen interactions (Arteca, 1996).
Gibberellins influence various developmental processes, including stem elongation, germination, dormancy, flowering, sex expression, enzyme induction, and leaf and fruit senescence. Gibberellic acid 3 (GA 3 ) helps in promoting plant growth and cell elongation and also stimulates the cells of germinating seeds to produce mRNA coding for hydrolytic enzymes.
Indole-3-acetic acid (IAA) is the main auxin in plants, controlling many important physiological processes including cell enlargement and division, tissue differentiation, and responses to light and gravity (Leveau, and Lindow, 2004). IAA and phenol compounds are known to play a key role in Fusarium wilt banana (Fernandez-Falcon et al., 2003).
Abscisic acid (ABA) is crucial in plant protection in a number of ways (1) it causes the stimulation of stomata closure (water stress brings about an increase in ABA synthesis); (2) inhibits shoot Correspondent Author: Mohamed M Ibrahim mohamednasr79@yahoo.com gibberellins on -amylase and has some effect on the induction and maintenance of dormancy. ABA also induces gene transcription particularly for proteinase inhibitors in response to wounding which may explain an apparent role in defense against pathogen attack (Davies, 1987;Hansen and Grosmann, 2000). ABA is also produced in the roots in response to stresses, including pathogen attack (Davies, 1987). ABA is involved in the wound-induced activation of the proteinase inhibitor II gene (Davies, 1987), a hypothesis compatible the model suggesting that this hormone is the mediator of the systemic wound response in tomato and potato (Gemishev et al., 2005). The role of ABA on plant resistance pathways is more complicated. ABA has been implicated in both biotroph and necrotroph susceptibilities. Therefore, it seems that this hormone acts as a negative regulator of plant defense. In addition, ABA plays a crucial role in response to abiotic stress. The two opposite roles of ABA on biotic and abiotic stresses lead to the hypothesis that plants prioritize the response towards abiotic stress over the response towards biotic stress. It is worth noting that ABA has also been associated with disease resistance. However, our understanding of its role in defense is rather limited (Lopez et al., 2008).
Plants respond to pathogen attack via a complex and integrated set of responses encompassing both constitutive and induced factors. Interactions between plants and pathogens initiate a series of events in plants, including production of signaling compounds such as ethylene, salicylic acid, jasmonic acid, and/or reactive oxygen species (Dowd et al., 2004). The response also clearly involves gene activation in the host plant, sometimes at sites remote from the point of wounding or infection (Pēna-Cortés et al., 1989) Disease affects plant growth and development of the plant by impacting on, amongst other regulatory processes, the PGRs through several possible actions including interfering with synthesis, degrading the PGRs or producing the PGRs directly (Agrios, 1997). The nature and rate of PGR production by a pathogenic micro-organism, however, may differ in vitro compared to in vivo. Information on the abilities of pathogens to produce PGRs under in vitro conditions, therefore, may give little insight into the situation occurring within the diseased plant (Agrios, 1997).
Some fungal pathogens are able not only to induce increasing IAA synthesis in their respective hosts, but also to produce IAA which may be directly released into the soil (Agrios, 1997). When plants encounter an invading pathogen, not only responses signaled by defense hormones are activated to restrict pathogen invasion, but also the modulation of additional hormone pathways is required to serve other purposes, which are equally important for plant survival, such as re-allocation of resources, control of cell death, regulation of water stress, and modification of plant architecture. Notably, pathogens can counteract both types of responses as a strategy to enhance virulence (Lopz et al 2008).
Trichoderma species have long been recognized as biological control agents (BCAs) for the control of plant diseases and for their ability to increase plant growth and development. These fungi are known to be involved in complex interactions with host plants and resident microbial communities. The mechanisms involved in the antagonism of Trichoderma toward phytopathogenic fungi include competition, antibiosis, direct mycoparasitism, and induction of systemic resistance to pathogens in planta. . These activities are related to plant growth stimulation but, given the role of PGRs in plant growth and development, it is pertinent to determine the impacts of beneficial microorganisms on concentrations of these substances in plant. The aim of the work reported here, therefore, was to determine the effects of T. harzianum and A. migulanus on PGR concentrations in Gladiolus grandiflorus.

Preparation of pathogen and antagonist inoculate
The isolate of F. oxysporum f. sp. gladioli, G010 was maintained on PDA (Oxoid, Basingtoke, Hants, UK) at 22° C and routinely sub-cultured at 15 day intervals.
Subcultures were prepared by inoculating PDA with 1 cm diam. disks of colonized PDA plus mycelium, cut from the edge of an actively growing, 7 day old colony.
Trichoderma harzianum isolate T22, obtained from CBS, was cultured on PDA. Petri dishes were sealed with Parafilm, and incubated at 22° C, with routine sub-culturing at 15 day intervals. Spore suspensions were obtained by flooding 7 day old cultures on PDA with 5 ml sterile distilled water, gently agitating the surface with a wire loop and passing the suspension through two layers of washed sterile muslin cloth directly into 50 ml centrifuge tubes. Spores were centrifuged at 1700 x g in a Thomson-MSE Mistral bench top centrifuge for 10 min, the spore pellets rinsed twice in sterile distilled water, with repeated centrifuging and spore concentrations adjusted to 8 ×10 6 ml -1 using repeated haemocytometer counts.
The isolate of A. migulanus was obtained from laboratory stocks and maintained on nutrient agar (NA; Oxoid, Basingstoke, Hants, UK) at 35° C, with routine sub-culturing at 15 day intervals. Cultures of A. migulanus were prepared by transferring approximately 1 ml of cell suspension from a 24 hour old liquid culture in tryptic soya broth (TSB; Oxoid, Basingstoke, Hants, UK) to 20 ml fresh TSB in 250 ml conical flasks. Flasks were incubated at 37 o C on a rotary shaker at 150 rpm for 24 h. The suspension was centrifuged at 1700 x g for 10 min, resuspended in 15 The future Journal of Biology 2019;3:44-53 ISSN:2572-3006(Print)2572-3111(Online) http://www.futurejournals.org ml quarter-strength Ringers solution (Sigma, UK) and washed 3 times in fresh Ringers solution by recentrifuging, as described above. Spore densities were estimated as colony forming units on NA following by serial dilutions to 5 × 10 8 .

Pathogen inoculation
Gladiolus corms were inoculated F. oxysporum f. sp. gladioli by removing 10 mm-diam. and 5 mm-depth piece of tissue from the side of the corm and replacing with a plug of inoculated PDA + fungal mycelium of the same dimensions, this method was modified from Cappelli and Minco (1998). The lesion area was calculated based on the height and width of the lesion. The lesion criteria were determined by the rotting and softening of the corm.

Antagonist inoculation
T. harzianum or A. migulanus were inoculated according to a modified method from (Elad et al., 1982). Ten corms were prepared for each treatment. Corms were submerged in T. harzianum, A. migulanus spore suspensions for 30 min. For the interaction treatments, corms were suspended in the antagonist suspension, blotted dry on sterilized filter paper under aseptic conditions in a laminar flow cabinet. The combination between T. harzianum and A. migulanus was prepared by mixing the same volume of suspension of antagonist in beaker, immersing surface sterilized corms in the mixed suspension for 30 min and inoculating with the pathogen, as described above. Control corms were immersed in sterilized distilled water for the same time. Inoculated corms were incubated at 25° C.

Corms inoculation
Gladiolus corms were inoculated under aseptic conditions and incubated at 25° C. Five replicated from each treatment were collected after 2 days of inoculation and the samples were collected at 2 days intervals.

Extraction of Plant growth regulators
Extractions of plant growth regulators from the infected Gladiolus corms were performed with modified methods of Yeang et al. (2001) and Durgbanshi et al. (2005). Briefly, 5 replicated of the treated corms were collected at 2-day intervals for 10 days. The tissue was frozen at -80° C until required. Frozen tissues were homogenized in liquid N 2 using a chilled mortar and pestle. The weight of each replicate was adjusted to be 20 g/ replicate. The weighted and homogenized tissue were added to 250 ml glass flasks and three volumes of dichloromethane (50 ml) were added and the extract sonicated for 30 min using a Bransonis 2510 sonicator (Branson Co., Danbury, USA). The extract was filtered twice through muslin cloth and the organic phase separated from the aqueous phase in a 100 ml separatign funnel. The pH of the organic phase was adjusted to 3.5 using 1N HCl and dried using a rotary evaporator (Buchi Rotavapor R-200, Switzerland) at 40 ○ C. The resulting residue was re-dissolved in 1.0 ml of methanol and kept frozen at -20 ○ C until analysis.

Liquid chromatography-mass spectrometry (LC-MS)
One sample from each treatment was subjected to (LC-MS) was obtained from a Thermo Instruments MS system (LTQ XL/ LTQ Orbitrap Discovery) coupled to a Thermo Instruments HPLC system (Accela PDA detector, Accela PDA autosampler and Accela Pump). The following conditions were used: capillary voltage 45 V, capillary temperature 260 °C, auxiliary gas flow rate 10-20 arbitrary units, sheath gas flow rate 40-50 arbitrary units, spray voltage 4.5 kV, mass range 100 -2000 amu (maximum resolution 30000).

High Performance Liquid Chromatography
High performance liquid chromatography (HPLC) was carried out on a C18 HD analytical column (250 mm x 4 mm; Agilent, USA) using an Agilent Series 1100 LC pump, connected to a Jasco MD-910 multiwave length detector (Jasco International Co. Ltd.) at room temperature. Samples (200 μl) were eluted in a linear gradient of 20 -80% acetonitrile and water acidified with 0.1% trifluoroacetic acid (Sigma-Aldrich) over 30 min..GA 3 , ABA, IAA were detected by monitoring absorbance of the eluate at 208, 265 and 280 nm, respectively, at a flow rate of 1 ml/ min.T he retention times of GA 3 , IAA, ABA were 12, 17 and 21 min, respectively.

Quantitation of Plant Growth Regulators
To prepare calibration curves, a modified method of Chiwocha et al. (2003) was used. PGRs were dissolved in methanol to prepares stock solution. 100 mg of each PGRs were dissolved in 1 ml methanol to prepare stock solutions for standard curve calibration. in to 5-ml volumetric flasks. The standard solutions were made up to 5 ml with 1:1 (v/v) acetonitrilewater containing 5% v/v glacial acetic acid. Each of the five standards was dried using a continuous air flow. The standards were reconstituted in 1000 µl methanol, sonicated and centrifuged at 1700 x g in a Thomson-MSE Mistral bench top centrifuge for 10 min. Aliquots of(20, 50, 100, 150, and 200 µl) of each PGR was injected into the HPLC system and the peak area was calculated automatically by Agilent 1200 software. Calibration curves were created for the three PGRs by plotting concentration against peak area.

Statistical analysis
Experiments were organized in complete block designs. Statistical analyses were conducted using the general linear model procedures of SPSS version 15. Experiments were analyzed using analysis of variance (ANOVA). Significance was evaluated at P<0.05 for all tests. Mean separation was tested using the Tukey HSD test.

RESULTS
GA 3 concentrations increased linearly after inoculation of corms with T. harzianum and A. migulanus. In contrast, corms inoculated with F. oxysporum f. sp. gladioli contained significant less in GA 3 compared with other treatments (Figure, 5.1; P< 0.001). Corms treated with both T. harzianum and A. migulanus had significantly higher contents of GA 3 . Between days 2 and 8, GA 3 concentrations in corms treated with T.harzianum or A. migulanus, followed by inoculation with F. oxysporum f. sp. gladioli were significantly higher than in corms treated with F. oxysporum f. sp. gladioli alone, or in control corms (P< 0.001). The GA 3 concentrations in corms treated with A. migulanus followed by inoculation with F. oxysporum f. sp. gladioli, however, were significantly lower than in those inoculated with A. migulanus alone (P>0.05). Corms treated with both antagonists prior to inoculation with F. oxysporum f. sp. gladioli had a significantly higher GA 3 concentration than those treated with A. migulanus alone (P<0.001).

DISCCUSION
This is the first paper to report the impacts of the application of Trichoderma harzianum and/or Aneurinobacillus migulanus on the concentrations of plant growth regulators in host plant tissues. Most previous work on the use of Trichoderma spp. as a biological control agent has concentrated on its efficiency as an antagonist against soil-borne and aerial pathogens (e. g. Elad et al., 1981; Elad, 2000), without considering the effects on host plant physiology. Work on A. migulanus has focused on its ability to control pathogens of aerial plant parts, such as mildews and grey mould (Edwards, and Seddon,

1992).
Knowledge of the actual mechanisms involved in the reduction of disease incidence following corm treatment with Trichoderma spp. is still incomplete. In the past, most studies have focused on microbial interactions in the rhizosphere, not on the possible impacts of the BCAs on the host plant, although increased plant growth response with penetration of T. harzianum into the root system has been demonstrated (Yedida et al., 1999). The results of the present work support the previously reported mechanisms responsible for the influence of Trichoderma treatments on plant development, such as the production of plant growth regulators (Inbar et al., 1994). Additional mechanisms of action include an improvement in the solubility of minor nutrients in Trichoderma-treated soils (Harman and Bjorkman, 1998), which can support an increase in the uptake and translocation of less available minerals and improvements in plant growth. Also, this work results support the findings of O 'Donnell et al., (2003), and Schmelz et al., (2003) when quantified the hormone content in Arabidopsis after challenge with two virulent pathogens: Xanthomonas campestris pv. campestris and Pseudomonas syringae pv. tomato DC3000 (PstDC3000). They found an increase of the usual stress hormones such as SA, JA and ET, they found rapidly elevated levels of auxin and induced auxin biosynthetic genes in comparison with mockinoculated plants. They showed that in the same conditions, the same pathogen also induced abscisic acid (ABA). The induction of auxin or auxin-related phenomena in a susceptible interaction is not an isolated case or a consequence of plant cell disorder.The ability of A. migulanus on increasing Trifolium repens L. growth and inhancing the plant Cd tolerance. Indole acetic acid produced by B. brevis may be related to its ability for improving root growth, nodule production and AM fungal intra and extraradical development ABA has also been associated with disease resistance. ABA-deficient mutants have been found to be insensitive to b-aminobutyric acid (ABA)-induced resistance. The mechanism by which ABA induces resistance against a pathogen is still unclear.ABA treatment mimicked the priming effect of BABAinduced resistance (Ton and Mauch-Mani, 2004). One possible explanation for the results obtained in this chapter and in chapter 8 is the timing of the infection. ABA controls stomatal closure and therefore controls the entry of the bacteria into the plant. Inside the plant, pathogenic bacteria need to maintain a high water potential in order to establish disease. The other well established role of ABA in water stress conditions or diseases conditions may also explain the effect of this hormone on plant susceptibility (Ton and Mauch-Mani, 2004). de Torres-Zabala et al., (2007) showed that bacterial effectors delivered into plant cells enhanced susceptibility by increasing ABA production and activating of ABA-responsive genes. In these studies ABA enhanced bacterial growth by attenuating basal defense, and ABA induction and suppression of basal defense transcripts could be mimicked by the in planta expression of the bacterial effector avrPtoB. ABA may thus have different actions at different infection steps, favoring resistance during pre-invasion, and susceptibility at later stages of colonization.
The network of interactions between hormones cannot be overlooked to fully understand the tight interaction between a host and its pathogen. Often a coordinated range of hormones is necessary to achieve the proper response (Schenh et al., 2000). Nemhauser et al., (2006) found the time course of the experiment will probably dictate the hormonal response observed and that could be a reason in this study. Pathogen-induced hormone disruption should be viewed as an imbalance in a network where every component will be affected, rather than as an interaction between two pathways. Much work is needed to understand the impact of each hormone on the disease pathway and to discover the nodes where the interactions occur The current work used plants grown in a soilless culture system, where soluble nutrients were provided in a recycled solution. The results of this study, however, have furthered understanding of how treatment with T. harzianum (and A. migulanus) enhanced growth of Gladiolus. But the mechanisms of action of these BCAs in this respect require further investigation, including work on the relationship between the effect of F. oxysporum f. sp. gladioli, T. harzianum, and A. migulanus secondary metabolites on PGR production by Gladiolus.