Production of Primary Metabolites by Rhizopus stolonifer, Causal Agent of Almond Hull Rot Disease

Species in the fungal genus Rhizopus are able to convert simple sugars into primary metabolites such as fumaric acid, lactic acid, citric acid, and, to a lesser extent, malic acid in the presence of specific carbon and nitrogen sources. This ability has been linked to plant pathogenicity. Rhizopus stolonifer causes hull rot disease in almonds, symptoms of which have been previously associated with the fungus’s production of fumaric acid. Six isolates of R. stolonifer taken from infected almond hulls were grown in artificial media amended with one of four carbon sources (glucose, fructose, sucrose, and xylose) and two nitrogen sources (asparagine and ammonium sulphate) chosen based on almond hull composition and used in industry. Proton nuclear magnetic resonance ((1)H NMR)–based metabolomics identified that R. stolonifer could metabolise glucose, fructose, sucrose, and to a lesser extent xylose, and both nitrogen sources, to produce three metabolites, i.e., fumaric acid, lactic acid, and ethanol, under in vitro conditions. Sugar metabolisation and acid production were significantly influenced by sugar source and isolates, with five isolates depleting glucose most rapidly, followed by fructose, sucrose, and then xylose. The maximum amounts of metabolites were produced when glucose was the carbon source, with fumaric acid produced in higher amounts than lactic acid. Isolate 19A–0069, however, preferred sucrose as the carbon source, and Isolate 19A–0030 produced higher amounts of lactic acid than fumaric acid. This is the first report, to our knowledge, of R. stolonifer producing lactic acid in preference to fumaric acid. Additionally, R. stolonifer isolate 19–0030 was inoculated into Nonpareil almond fruit on trees grown under high– and low–nitrogen and water treatments, and hull compositions of infected and uninfected fruit were analysed using (1)H NMR–based metabolomics. Glucose and asparagine content of uninfected hulls was influenced by the nitrogen and water treatments provided to the trees, being higher in the high–nitrogen and water treatments. In infected hulls, glucose and fructose were significantly reduced but not sucrose or xylose. Large amounts of both fumaric and lactic acid were produced, particularly under high–nitrogen treatments. Moreover, almond shoots placed in dilute solutions of fumaric acid or lactic acid developed leaf symptoms very similar to the ‘strike’ symptoms seen in hull rot disease in the field, suggesting both acids are involved in causing disease.