Soybean, the most important agricultural product in Brazil, is widely cultivated all over the country. The occurrence of green stem and foliar retention (GSFR) has been reported since the beginning of the soybean cultivation in Brazil and its potential causes were attributed to severe attack of stinkbugs or plant nutritional disorders. About two decades ago, a new type of GSFR was reported in Brazilian tropical regions of soybean production, also of an unknown cause. Several possible causes were investigated, but the presence of the nematode Aphelenchoides sp. in symptomatic plants was frequently observed. Koch's postulates were conducted to check whether this nematode could be the cause of the syndrome. Specimens of Aphelenchoides sp. were isolated from soybean plants and multiplied in colonies of the fungus Fusarium sp. in PDA culture medium. Nematode suspensions were inoculated onto healthy soybean plants and the typical GSFR symptoms were observed. The nematode was recovered from the inoculated plants. Based on both morphological and molecular data, we suggest that Aphelenchoides besseyi is the causal agent of GSFR of soybeans in Brazil, a syndrome popularly known as "Soja Louca II".

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Rationale The nematode Aphelenchoides besseyi is the causal agent of green stem and foliar retention, a soybean disease recently described in Brazil. This condition can reduce soybean yield by up to 100%. However, little is known about chemical interactions between the plant and pathogen. Therefore, this work aimed to investigate metabolites from healthy soybean roots and from soybean roots that were inoculated with A. besseyi. Methods A. besseyi were multiplied in vitro with Fusarium sp. colonies in Petri dishes for 25 days, and were axenically inoculated into hydroponics healthy soybean plants. The metabolites were extracted from the roots of healthy and A. besseyi‐infected plants 16 days post‐inoculation. These extracts were analyzed using an untargeted metabolomic method with an ultra‐high‐performance liquid chromatography/electrospray ionization /tandem mass spectrometry (UHPLC/ESI‐MS/MS) and molecular networking approach. Results Roots from infected plants showed morphological alterations such as shrinkage, darkening, and arching. Similarly, they also showed an increased presence of flavonoids, compared with healthy roots. Compounds such as neobavaisoflavone, glycitin, genistin, and genistein were putatively identified and had greater intensity in inoculated roots. These compounds are linked to the defensive mechanisms in plants against nematodes. Moreover, coumaric acid, also exclusively putatively identified in inoculated roots, shows activity related to inhibition of root growth. Conclusions Liquid chromatography, mass spectrometry, and molecular networking approaches proved to be a powerful tool for the metabolomic study of GSFR. This study showed metabolomics differences of protective substances in the roots, evidencing a quick response of the plant to the attack of A. besseyi.