Effect of Indole-3-Acetic Acid and Flavonoids on Association of Maize Roots and Phytostimulant Endophytic Fungi

Chemical communication between microbes and their host is the first step toward the establishment of any association. Plant roots release a variety of signaling molecules that naturally screens the surrounding microbes for association. Current study focuses on isolation of IAA and flavonoids producing endophytic fungi and the influence of these metabolites on root-endophyte interaction. For this purpose, 16 endophytic fungi were isolated from Asphodelus tenifolius and Withenia somnifera collected from the dry areas of district Malakand. Based on screening, for their efficiency to produce metabolites (IAA, ammonia and flavonoids) used in plant growth, five endophytes were selected for further study including their identification by sequence homology of the internal transcribed spacer (ITS) region of 18 S rDNA, their plant growth promoting potential, identification of phenols and flavonoids produced by these strains alone or in co-culture with maize and their colonization in maize root under different treatments. The selected strains including wl1, wlg2 and greenish were identified as species of Aspergillus awamori, Aspergillus nomius and Aspergillus fumigatus while two strains (Pz and wlw) belonges to Fusarium culmorum and Fusarium oxysporum. The selected endophytes produced significant amount of IAA ranging between 24.2 and 86.4 µg/mL where maximum production recorded in Aspergillus nomius wlg2. The most efficient flavonoids producing strains were Fusarium culmorum Pz and A. nomius wlg2 with 275 and 261 µg/mL flavonoids respectively in their cultures. LC-ESI-MS/MS analysis of the culture filtrate resulted in the identification of 3 flavonoids (calycosin, dihydroxyflavone and pratensein) from Fusarium culmorum strain Pz and 5 flavonoids (4-bromo 3nitroacetophenone, dihydroxyflavone, apigenin-6,8-di-c-glycoside, rutin, 3hydroxyphelorutin,3,5-di-c-hexoside from A. nomius wlg2. Production of flavonoids and fungal growth were significantly compromised when the endophytes were exposed to 100 µL of cinnamic acid (CA). Interestingly, the CA exposed cultures had only one species of flavonoids indicating the influence of this compound on flavonoids biosynthesis. For instance, CA exposed cultures of F. culmorum and A. nomius had only dihydroxyflavone and apigenin6,8-di-c-glycoside respectively. Phenols were also produced by the isolatesin a concentration ranging from 303.8 µg/mL (A. awamori w11) to 617.6 µg/mL (A. nomius wlg2). Phenolic acid, chlorogenic acid was identified in cultre filtrate of A. nomius wlg2 whereas two phenolic acids including cumaric acid and caffoly-d- glucoses were identified in culture filtrate of endophyte F. culmorum Pz using LC-ESI-MS/MS.All the selected strains significantly improved growth parameters viz shoot length, root length, fresh and dry weight of maize seedlings when treated with endophyte culture filtrate or grown in co-culture with the endophyte under hydroponics. F. culmorum Pz and A. nomius wlg2 showed the best results in culture filtrate treated maize seedlings. In co-culture experiment maximum increase of shoot and root length (33% and 60% respectively over control) corresponding to A. nomius wlg2 and Fusarium wlw.Exogenous application of IAA and GA3 significantly improved growth of maize seedlings under hydroponics. Exogenous IAA significantly promoted maize root colonization by all the isolates and increased shoot length. Additionally, the rate of colonization was higher in zone of division of root in all IAA treated seedlings. Maximum promotion in shoot length (53%) was recorded in seedlings hosted endophyte A nomius wlg2 or F. oxysporum wlw in addition to foliar IAA application. Contrary to this, GA3 promoted seedling growth but had no positive role like IAA in root colonization by endophytes The maize seedlings released significant amount of flavonoids, phenols and sugars in their root exudates. Three different types of flavonoids (Quercetagatin, rhamnocitrin and 7,2,dihydroxy-4-methoxyl isoflavan) were detected in root exudate of control maize seedlings, however, only quercetagtin was present in exudates of CA treated seedlings. Quality and quantity of flavonoids released from maize root were influenced by the endophytes and also the maize associated endophytes showed a change in flavonoids profile. For instance, the coculture of maize seedlings and endophyte Fusarium culmorum Pz had quercetagtin and rhamnocitrin and calycosin indicating a clear qualitative change in the flavonoids profile. Likewise apigenin-6,8-di-c-glycoside, letuloin, calycosin, ramnocitrin and quercetagatin were detected in root exudates of seedlings associated with wlg2. These results are quite interesting because letuloin and calcyosin were neither present in exudates of control nor in that of wlg2 colonized seedlings. The new flavonoids might have a role in plant-endophyte interactions.CA or yucassin treatment arrested shoot growth, however normal growth (or even better than control in case of wlw) was observed in endophytes associated seedlings. Root growth and seedling biomass was also negatively influenced by CA or yucasin treatment however endopytes associated seedlings supressed the activities of CA or yucasin and seedlings have normal roots and biomass (or even better than control in case of F. oxysporum wlw, A. nomius wlg2 and F. culmorum Pz). All the isolates were capable to colonize maize roots where wlg2 was the most efficient one. CA and yucasin inhibited biosynthesis of flavonoids and IAA by the plants accompanied by reduced efficiency of endophytes to colonize maize roots. CA and yucasin inhibited biosynthesis of flavonoids and IAA by the plants accompanied by reduced efficiency of endophytes to colonize maize roots. However foliar CA or yucasin did not affect exudation of sugars but seedlings associated with endophytes and received these inhibitors improved exudation of sugars. Interestingly, flavonoids, letuloin and calcyosin were neither present in exudates of control nor in that of wlg2 colonized seedlings. Fungal flavonoids calycosin and apigenin-6,8-di-c-glycoside, and maize flavonoids ramhnocitrin andquercetagtin were involved in root endophyte interaction. To conclude, this work reveals that plant root-fungal endophyte association is animated by a complex chemical dialogue between both the partners where IAA, phenols, flavonoids and sugars are among the key players. The study also urges for further investigation to completely understand the mechanism by exploring the exact role of each component of the dialogue leading to successful association.