THE INFLUENCE OF SPRAYING WITH POTASSIUM SILICATE AND IRRIGATION WITH SALINE WATER IN SANDY SOIL ON Calendula officinalis L.

Two pots experiments were conducted at the Experimental Farm of El-Qassasin Horticultural Research Station, Agricultural Research Center, Ismailia Governorate, Egypt, during two successive seasons of 2016/2017 – 2017/2018, to investigate the effect of potassium silicate at (0, 4, 6, and 8 cm3/l) as a foliar spray under different levels of water salinity (tap water, 1000, 2000 and 3000 ppm) on Calendula officinalis L. plant. The experiment was performed in complete randomized block design as factorial experiment with 3 replicates. The obtained results cleared that using salinity levels decreased growth parameters (plant height, number of branches/plant, fresh and dry weights of herb/plant), flowering parameters (flower diameter, number of flowers/plant, fresh and dry weight of flowers (g/plant) during eight cuts and fresh and dry weight of flowers (g/plant/season)) and chemical constituents (chlorophyll a, chlorophyll b, carbohydrate and carotenoid contents) compared to control. Moreover, the highest values in these parameters were registered by potassium silicate at 8 cm3/l concentration. Proline content increased in leaves with using saline water at 3000 ppm + potassium silicate at 8 cm3/l. Generally, it could be concluded that potassium silicate at 8 cm3/l, showed a uniform impact in alleviating inhibition of Calendula officinallis L. plant growth and productivity under moderate salinity stress condition.


INTRODUCTION
Hydroponic culture became the new modern agricultural technology this years; however the spreading of pathogens considers the major problem especially in closed system technique (Nosir, et al 2009). In recent years there has been growing interest in the potential use of microbial metabolites as agrochemicalsas an alternative to chemical fungicides. Microbial metabolites may help overcome problems associated with resistance of pathogenic fungi to fungicides and are generally more biodegradable and environmentally friendly than their synthetic counterparts (Tanakaand Mura, 1993). A wide range of antibacterial and antifungal secondary metabolites has been characterised from fungi, including from Trichoderma spp. (Vey et al., 2001). The correlation between the production of antimicrobial metabolites by biological control agents and the effectiveness of Biological control agents (BCA) preparations in vivo is still a matter of conjecture. Secondary metabolites are a heterogeneous group of natural compounds that are considered to aid the producing organism in survival and basic functions, such as competition, symbiosis, metal transport and differentiation (Demain and Fang, 2000). Secondary metabolites secreted by Trichoderma spp. include volatile and non-volatile antifungal substances, such as 6-n-pentyl-6H-pyran-2one (6PP), gliotoxin, viridin, harzianopyridone, harziandione and peptaibols (Reino et al., 2008). The activities of these metabolites against soilborne plant pathogens have long been studied (Ghisalberti and Sivasithamparam, 1998). Production of secondary metabolites is considered an important factor in biological control, although the mechanisms of action of these compounds in soil and plants are not yet fully elucidated (Demain and Fang, 2000).

Species of
The involvement of secondary metabolites produced by Trichoderma spp. in the activation of plant defence mechanisms and the regulation of plant growth was recently investigated, using tomato and oil-seed rape seedlings treated with harzianolide and 6PP isolated from T. harzianum, followed by infection with spore suspensions of Botrytis cinerea and Leptosphaeriamaculans, respectively. In both host plant species, a reduction in disease symptoms was observed, particularly on 6PP-treated plants. Moreover, application of the metabolites lead to overexpression of pathogenesis-related (PR) proteins in treated plants (Vinale et al., 2008).

Antibiosis
assays demonstrated that secondary metabolites produced by T. harzianum (azaphilone, butenolide, harzianolide, harzianopyridone) had different activities towards Rhizoctoniasolani, Pythiumultimum and Gaeumannomycesgraminis var. triticiin in vitro tests, suggesting that individual compounds had specific modes of action (Vinale et al, 2006). The level of production of metabolites by Trichoderma spp. varies according to the target pathogen and the strain of Trichoderma in use. Increased concentrations of 6PP were secreted by T. harzianumin interactions with Botrytis cinerea, but the absolute concentration produced was related to the isolate of Trichoderma used (Cooney and Lauren, 1998). There are no reports in the literature on the effects of F. oxysporum f. sp. gladioli on T. harzianum natural products.
The aims of the work presented here were to develop an improved understanding of the roles of major secondary metabolites produced by T. harzianum in the interaction between T. harzianum,F. oxysporum f. sp. gladioli.  10 H 14 O 2 , and thus 4 degrees of unstauration. The 13 C NMR spectrum indicated a pyrone carbon at δ163.0, an oxygenated carbon at δ167.0, 3 olefinic carbons (δ105-140), 4 methylene carbons (δ20-35) and one methyl carbon at δ14.0 (Figure 2 &3). The UV spectrum of compound 1 had a peak at 336 nm ( Figure  4). Database searches indicated that the major unknown compound was 6-n-pentyl-6H-pyran-2-one (6PP). 6PP was the most secondary metabolite in liquid cultures of T. harzianum, with a yield of 0.356 mg.l -1 .

Isolation and chemical characterization of major compounds
The mass spectrum of compound 2 showed a molecular ion [M] + at m/z 366.19 ( Figure 5) corresponding to the molecular formula C 19 H 27 NO 6 . The structure of compound 2 was deduced from 1 H NMR and 13 C NMR spectral data ( Figures 6 and 7). The 1 H NMR in CD 3 OD exhibited a doublet at 7.09 ppm (2-H, 7=15Hz), an olefinic proton at 7.53 ppm (3-H) and overlapping signals cantered at 6.39 ppm (4-H and 5-H). Analysis of the 13 C NMR spectrum of compound 2 suggested that compound 2 had a carboxyl group (178.6 ppm). The UV spectrum of harzianic acid had a peak in absorbance at 259 nm ( Figure 8). Database searches and direct comparison with mass and 1 H and 13 C NMR spectral data identified the compound as harzianic acid.

Production of 6PP and harzianic acid in liquid cultures
T. harzianum produced 0.085 mg/ ml 6PP in the liquid medium . In dual cultures with T. harzianum and F. oxysporum f. sp. gladioli, however, 0.125 mg/ml was recovered. Harzianic acid concentrations were nearly doubled in dual cultures of T. harzianum and F. oxysporum f. sp. gladioli, based on peak heights recorded on HPLC.

Production of 6PP and harzianic acid in Gladiolus corms
In gladiolus corms treated with T. Harzianum alone, 0.058 mg.g -1 6PP was found; in contrast, corm tissues treated with T.harzianum followed by inoculation with F. oxysporum f. sp. gladioli had 6fold greater concentrations of 6PP (0.354 mg.g -1 ) were observed Harzianic acid concentrations were nearly double in T. harzianum-treated and F. oxysporum f. sp. gladioli inoculated corms, compared with corms treated with T. Harzianum alone. Concentrations of HA were estimated based on peak heights recorded on HPLC.

Biological activity of 6 PP
F. oxysporum f. sp. gladioli growth was completely inhibited by 6PP at all concentration from 10 µg to 100 µg on PDA in Petri dishes.

Effect of 6PP on Gladiolus growth in Hydroponic cultures
Gladiolus plant that coated by 10ppm of 6PP germinated 3 days earlier than the untreated and the flower harvest yield was 35 % more than the control in flower fresh weight. Flower stalk diameters in the treated corms with 6PP were 29.5 % taller than the control. The bullet numbers were 46 % more than the untreated. (data not shown).

DISCCUSION
The work reported here confirmed the production of antibiotic compounds by T. harzianum within corm tissues of Gladiolus infected with F. oxysporumf.sp. gladioli. Hiterhto, the ability of Trichodermaspp to secrete antibiotic compounds in infection courts had not been confirmed (Whipps, 2001). Distinguishing the antibiotics secreted in the presence of fungal diseases will help in understanding the mechanisms of action utilized by T. harzianum in the biological control of plant pathogens.  The aim of this work was to develop further understanding of the roles of secondary metabolites produced by T. harzianum during intereactions with F. Oxysporum f.sp. gladioli in interactions with corm tissues of Gladiolus grandiflorus tissues. The role of antibiotics produced by Trichoderma in biological control remains a matter of conjecture. Although certain metabolites with antibiotic activity may be major factors in the biocontrol activity of a given isolate of the fungus, this may not be the case for other isolates (Harman 2000). The detection of 6PP as the most abundant antifungal compound produced by T. harzianum T22 in the presence or absence of F. oxysporum f. sp. gladioli suggest that this metabolite may be of interest in terms of bio-fungicide potential. Similar results were reported by Vinale et al. (2008), who recorded 6PP as a major secondary metabolite secreted by T. harzianum, with activity against B. cinereaon tomato and L. maculatnson oil seed rape. The results obtained in the present work support the suggestion that antibiotic production during saprophytic and antagonistic growth of T. harzianum could be involved, in concert with other mechanisms, in the inhibitory interaction with plant pathogens (Howell, 2004).
F. oxysporumf. sp. gladioli showed greater sensitivity to 6PP than previously reported for Rhizoctoniasolaniand a different F. oxysporum (Scarselletti and Faull, 1994). In the earlier work, addition of 300 µg/ ml 6PP to Potato Dextrose medium caused a 69.6% growth reduction in R. solani and a 31.7% reduction in F. oxysporum after 2 days, and completely inhibited the germination of Fusarium spores at a concentration of 450 µg/ml. Although, B. cinerea metabolized 6 PP in agar and on liquid cultures (Cooney and Lauren, 1998). Moreover, a strong relationship was found between the production of 6PP and the antagonistic ability of by T. harzianum in vitro and control of B. cinerea rots in stored kiwi fruits has also been investigated by Poole et al. (1998).
This work findings described in this manuscript confirms Vinale et al., (2008) findings which reveal that the secondary metabolite, 6PP excreted by the antagonistic fungus T. harzianum not only interfered with the pathogenic fungus Botrytiscinerea or Leptosphaeriamaculans by inhibiting the mycelial growth and conidia production and germination but by promoting the plant growth and increased the plant resistance against diseases that could be one of the mechanisms T. harzianum used to stimulate plant growth. Ghisalberti and Sivasithamparam (1998) previously suggested that some secondary metabolites were directly involved in T. harzianum-plant interactions, and that the compound 6PP may act as an auxin-like compound and/or may act as an auxin inducer.
The chemical structures of the Trichoderma metabolites isolated in the present work suggests two different possible mechanisms of action. These low molecular weight, non-polar, 6PP and harzianic acid, are produced in high concentrations in the soil environment, and have a relatively long range of influence on the microbial community. In contrast, a short distance effect may results from the presence of polar metabolites and peptaibols acting in close proximity to the producing hyphae (Lorito et al.,1996).  This work findings and Vinale et al., (2009) findings help in opening a new way in studying the role of metabolites secreted from biocontrol agents in the glasshouse or field studies as the used of use of new products based on biocontrol agents and/or their metabolites for disease control is one of the most promising ways to reduce the dependence on synthetic pesticides in agriculture. Various biocontrol agents have been registered and are available as commercial products, including strains belonging to the genus Trichoderma but according to our knowledge there are no registered bio-products from trichoderma metabolites, although Fungal strains of the genus Trichoderma are well-known producers of secondary metabolites with antibiotic activity. Their production varies in relation to (i) the specific compound; (ii) the strain and the species; (iii) the presence of other microbes; and (iv) the balance between elicited biosynthesis and biotransformation rtes (Harman, 2004).
Understanding of the roles played by the secondary metabolites of T. harzianum may further the development of new, targeted biopesticides and bio-fertilizers based on these naturally occurring compounds. Such compounds may be used as elicitors of plant defense mechanisms, direct toxins to the pathogens and plant growth stimulants. This is the first work to report the production of secondary metabolites by T. harzianum in Gladiolus corms infected with F. oxysporum f. sp. Gladioli in hydroponics. The results improve understanding of the interaction between T. harzianum and F. oxysporum f. sp. gladioli in the host plant tissues. Further studies on the range of antibiotic compounds produced by T. harzianum and the efficiency of these compounds in plant-pathogenantagonist inereactions are required to further increase understanding of the mechanism of action of these bioactive compounds under field conditions.

Preparation of pathogen inoculum
The inoculum was prepared using an isolate of F. oxysporum f. sp. gladioli isolated in this work from a purchased Gladiolus corm. The culture was maintained on PDA (Oxoid, Basingtoke, Hants, UK) at 22 o C and routinely sub-cultured at 15 day intervals. Subcultures of F. oxysporum f. sp. gladioli 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.

Preparation of antagonist inocula
Trichodermaharzianum isolate T22, used as a fungal antagonist in this study, was obtained as freezedried spores from CentraalbureauvoorSchimmelculturen CBS, The Netherlands.One ml sterilized distilled water was added to the freeze dried spores and 0.1 ml of spore suspension used to inoculate fresh PDA in 9 cm diam. Petri dishes. Cultures were sealed with Parafilm (Alpha Laboratories, Hampshire, UK)., and incubated at 22 o C with routine sub-culturing at 15 day intervals. Subcultures of T. harzianum 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.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 directly into 50 ml centrifuge tubes. Spore suspensions were centrifuged at 3000 rpm (1700 x g) in a Thomson-MSE Mistral bench top centrifuge for 10 min. Following rinsing the spore pellets twice in sterile distilled water, with repeated centrifuging (as above); spore concentrations were adjusted to 4 .00 ×10 8 spores ml -1 using repeated hemocytometer counts under a light microscope at a magnification of ×40.

Inoculation with antagonists
Gladiolus corms, variety Big flower GT01 size 14 (Tylore Bulb, Co., The Netherlands) were surface sterilized in 20 % NaOCl for 20 min before rinsing in running tap water for 6 hours, followed by 3 rinses in sterilized distilled water. Corms were submerged in T. harzianumor A. migulanus spore suspensions for 30 min. For the interaction treatments, corms were suspended in the antagonist suspension and gently blotted dry on sterilized Whatman, No 3 filter paper under aseptic conditions in a laminar flow cabinet. The combination between T. harzianum and A. migulanus was prepared by mixing equal volumes of antagonist suspensions in a 2000 ml beaker, immersing surface sterilized corms in the mixed suspension for 30 min and inoculating with the pathogen, as described below. Control corms were immersed in sterilized distilled water for the same length of time.

Inoculation with Fusarium oxysporumf. sp. gladioli
The Gladiolus corms inoculated with antagonists were subsequently inoculated with F. oxysporum f. sp. gladioli by removing a 10 mm diam., 5 mm deep piece of tissue from the surface of the corm and replacing it with a plug of PDA plus fungal mycelium of the same dimensions. The corms after inoculation were incubated in 22° in laminar flow Lesion areas developing on inoculated corms were measured 3 days after treatment to estimate the efficiency of the antagonists before collecting the samples for analysis.

Antagonist and pathogen culture
T. harzianum alone; T. harzianum + F. oxysporum f. sp. gladioliT. harzianum treatments (1 L each); was cultured and the secondary metabolites extracted using the method of Vinale et al., (2006;2008). Two 10 mm diameter plugs of T. harzianum T22 were taken from the actively growing margin of cultures on potato dextrose agar (PDA; Oxoid, Basingtoke, Hants, UK) cultures and inoculated into 250 ml conical flasks containing 100 ml of full strength potato dextrose broth (PDB; Oxoid, Basingtoke, Hants, UK).
The same technique was used in dual cultures of T. harzianum and F. oxysporum f. sp. gladioli, the previous method was used, with addition of two 10 mm diameter plugs of F. oxysporum f. sp. gladioli into the same flask with T. harzianum. Cultures were incubated on rotary shaker at 220 rpm (Gallenkamp, Rhys Scientific Ltd, UK) for 15 days at 22° C.

Inoculation of Gladiolus corms
Gladiolus corms were inoculated with T. harzianum and A. migulanus, followed by inoculation with F. oxysporum f. sp. gladioli. The inoculated corms were incubated over moistened filter paper (Whatman No 1) with sterilized distilled water in plastic containers (22× 12× 32 cm) at 22° C. The filter papers were moistened daily under aseptic conditions.

Extraction and quantitation of secondary metabolites
The inoculated corms were frozen in liquid N and homogenized into fine powder using a hilled mortar and pestle. Aliquots (20 g) of the powder were extracted exhaustively with ethyl acetate (EtOAc) at room temperature for 3 times. The combined organic fraction was dried over Na 2 SO 4 and evaporated under reduced pressure at 35° C. The residue was taken up in 5 ml of EtOAc and subjected flash column Fractions of 20 ml were collected and subjected to analytical TLC. Fractions showing similar TLC profiles based on UV detection at 254 nm were combined and purified compounds subjected to mass analysis using liquid chromatography-mass spectrometry (LC/MS; Thermo Instruments.) Further purification of the flash column chromatography residue from each treatment was carried out using high performance liquid chromatography (HPLC) on a C18 HD analytical column (250 mm x 4 mm; Agilent, USA), with an Agilent Series 1100 LC pump (Agilent, USA), coupled to an LC 90 UV spectrophotometer (Jasco International Co. Ltd.). Samples (100 μl) were eluted at room temperature in a linear gradient of 20 -80% acetonitrile in acidified water (0.1% trifluoroacetic acid; Sigma-Aldrich) over 30 min Purified compounds were characterized and identified using accurate mass analysis (LC/ MS). High resolution mass spectral data were obtained using 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).
Compounds were identified, based on MS and NMR data by comparison with the Beilstein database (2010) and directly with mass and 1 H and 13 C NMR spectral data in the literature (Dunlop et al., 1989). 1 H and 13 C NMR analysis were done on Varian 400MHz NMR spectrophotometer.
The filtered broth cultures of each treatment (1 L each); T. harzianum alone; T. harzianum + F. oxysporum f. sp. gladioli were extracted with ethyl acetate (3 x 500 ml each). Cultures were filtered under vacuum through Whatman No. 4 filter paper (Brentford, UK) and the filtrates stored at 2° C for 24 h. Filtrates were extracted three times in 500 ml EtOAc, organic phases combined and rotary evaporated (BuchiRotavapor R-200, Switzerland) to dryness under reduced pressure at 40° C. The residue obtained from T. harzianum was brown (1.5 g); T. harzianum + F. oxysporum f. sp. gladioli reddishbrown (0.869 g), and T. harzianum + F. oxysporum f. sp. gladioli + A. migulanus yellow-brown (0.475 g). Residues were re-dissolved in 3 ml methanol each and further purified by flash column chromatography, using isocratic elution in EtOAc: hexane (2:1 v/v), as described above.

Biological activity of 6PP
The purified 6PP was tested against growth of F. oxysporumf. sp. gladioli using the method of Vinaleet al. (2008). Discs of fungal mycelium on PDA, 10 mm in diam., were placed at the centre of 90 mm diam. Petri dishes containing fresh PDA. Test compounds were added in a 10 l drop of methanol containing 10, 20, 50 and 100 µg of the compound per plug of F. oxysporum f. sp. gladioli. Cultures were incubated at 25° C for 7 days. Pathogen growth was measured daily. Each treatment consisted of three replicates and the experiment was repeated twice.

Effect of 6PP on Gladiolus growth in Hydroponic cultures
Gladiolus corms were grown hydroponically as described by Nosir et al, 2009. The corms were treated with 10ppm solution of 6PP in Arabic gum and kept for drying at room temperature.