Characterization and Pathogenicity of Rhizoctonia solani Isolates Affecting Pisum sativum in North Dakota.

Acreage of dry field pea (Pisum sativum) in North Dakota has increased approximately eightfold from the late 1990s to the late 2000s to over 200,000 ha annually. A coincidental increase in losses to root rots has also been observed. Root rot in dry field pea is commonly caused by a complex of pathogens which included Fusarium spp. and Rhizoctonia solani. R. solani isolates were obtained from roots sampled at the three- to five-node growth stage from North Dakota pea fields and from symptomatic samples received at the Plant Diagnostic Lab at North Dakota State University in 2008 and 2009. Using Bayesian inference and maximum likelihood analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA), 17 R. solani pea isolates were determined to belong to anastomosis group (AG)-4 homogenous group (HG)-II and two isolates to AG-5. Pathogenicity of select pea isolates was determined on field pea and two rotation hosts, soybean and dry bean. All isolates caused disease on all hosts; however, the median disease ratings were higher on green pea, dry bean, and soybean cultivars when inoculated with pea isolate AG-4 HG-II. Identification of R. solani AGs and subgroups on field pea and determination of relative pathogenicity on rotational hosts is important for effective resistance breeding and appropriate rotation strategies.

Further analysis of maize C(4) pyruvate,orthophosphate dikinase phosphorylation by its bifunctional regulatory protein using selective substitutions of the regulatory Thr-456 and catalytic His-458 residues.

In C(4) plants such as maize, pyruvate,orthophosphate dikinase (PPDK) catalyzes the regeneration of the initial carboxylation substrate during C(4) photosynthesis. The primary catalytic residue, His-458 (maize C(4) PPDK), is involved in the ultimate transfer of the beta-phosphate from ATP to pyruvate. C(4) PPDK activity undergoes light-dark regulation in vivo by reversible phosphorylation of a nearby active-site residue (Thr-456) by a single bifunctional regulatory protein (RP). Using site-directed mutagenesis of maize recombinant C(4) dikinase, we made substitutions at the catalytic His residue (H458N) and at this regulatory target Thr (T456E, T456Y, T456F). Each of these affinity-purified mutant enzymes was assayed for changes in dikinase activity. As expected, substituting His-458 with Asn results in a catalytically incompetent enzyme. Substitutions of the Thr-456 residue with Tyr and Phe reduced activity by about 94 and 99%, respectively. Insertion of Glu at this position completely abolished activity, presumably by the introduction of negative charge proximal to the catalytic His. Furthermore, neither the T456Y nor inactive H458N mutant enzyme was phosphorylated in vitro by RP. The inability of the former to serve as a phosphorylation substrate indicates that RP is functionally a member of the Ser/Thr family of protein kinases rather than a "dual-specificity" Ser-Thr/Tyr kinase, since our previous work showed that RP effectively phosphorylated Ser inserted at position 456. The inability of RP to phosphorylate its native target Thr residue when Asn is substituted for His-458 documents that RP requires the His-P catalytic intermediate form of PPDK as its protein substrate. For these latter studies, synthetic phosphopeptide-directed antibodies specific for the Thr(456)-P form of maize C(4) PPDK were developed and characterized.