Role Of GDH2 Gene In Ammonia Production And Trap Formation In Arthrobotrys Oligospora Induced By Nematode Extract

The economic losses caused by plant parasitic nematodes worldwide are severe every year.Nematophagous fungi,which are widely concerned because of their variety and wide distribution,are the natural enemies of nematodes.Their popular model species Arthrobotrys oligospora produce three-dimensional trap that can prey nematode.The biological factors such as nematode,bacteria and other non-biological factors,such as amino acids and peptides,can induce Arthrobotrys oligospora to form traps.Many researches and our previous study revealed that A.oligospora secreted ammonia when it was cultured with nematode extracts and ammonia could induce trap formation in A.oligospora.Many fungi have been reported to secrete ammonia and alkalinize environmental pH.Alkalinized environment can activate fungal pathogenicity and the expression of virulence traits.There are many genes that were reported to associated with ammonia secretion.These genes are including AreA,Mep,PacC,GDH2,GS1,GDH2?coding glutamate dehydrogenase?and GS1?coding glutamine synthetase?,amet gene?coding ammonia transporters?.In this paper we studied the role of three genes GDH2,GS1 and amet in ammonia production and trap formation in A.oligospora by nematode extract?NE?.Three genes of GDH2,GS1 and amet were knocked out by the homologous recombination method,and only the GDH2 mutant was obtained.We compared the A.oligospora wild-type?WT?with GDH2 mutant strains in growth rate,spore production,growth resistance,ability to kill nematodes,trap formation by NE,alkalinization determined by pH fluorescent dye,metabolite differences by HPLC.There were no significant differences between A.oligospora wild-type?WT?with GDH2 mutant strains in the growth rate,growth resistance,and the ability of killing nematodes.There were significant differences between the HPLC profiles of the fermentations of WT and the GDH2 mutant.GDH2 mutants showed 50%reduced ammonia secretion,while exogenic ammonia addition restored 13%trap formation.GDH2 mutant strains of A.oligospora,impaired in ammonia production,show 90%inhibition in trap formation induced by NE.Microscopic observations of mycelia of the WT strain showed a strong internal fluorescence signals than GDH2 mutant strains,as detected by using the alkaline detector fluorescence dye BCECF/AM.The results suggested that GDH2genes play an important role in ammonia production and trap formation in A.oligospora.The spore production capacity of GDH2 mutant strains decreased by66.4%compared with WT,suggesting that the GDH2 gene played an important role in the spore production.In the medium containing 0.01%SDS,0.02%SDS,0.1M NaCl,0.2 M NaCl,5mM H₂O₂,10mM H₂O₂ and 15mM H₂O₂ there was no difference in the resistance of WT and gd mutant strain,but in the medium of 0.03%SDS and 0.3M NaCl,the resistance of the GDH2 mutant was less than that of WT.Some researches reported that volatile substances secreted by A.oligospora could attract nematodes,thus increasing the chance of killing nematodes.Using agarose gel plate method ammonia concentration between 10^-8 and 10^-2 attracted c.elegans?attract index>0.2?,the results suggest that ammonia can attract C.elegans.Highlights of this article:1.Our results suggest that the GDH2 gene of A.oligospora plays an important rolein ammonia production,trap formation and spore production.2.The study proved that ammonia could attract nematodes,which may increase theability of A.oligospora to kill nematodes.