University of Puerto Rico, Crops and Agro-Environmental Sciences, Plant Disease Clinic at Juana Diaz, PR
University of Florida, Institute of Food and Agricultural Sciences, Plant Pathology Department, Plant Diagnostic Center, Gainesville, FL
Sentinel plots for monitoring Asian soybean rust (ASR) caused by Phakopsora pachyrhizi Syd. were initiated in 2005 at Isabela (EEI), Adjuntas (EEA), and Juana Diaz (EEJD) experiment stations. Until 2009, no signs or symptoms of ASR were observed in soybean (Glycine max [L.] Merr.) or common bean (Phaseolus vulgaris L.). These sites were found to be negative for the occurrence of ASR based on PCR with specific primers Ppa1 and Ppa2 (2). However, P. meibomiae, the cause of American soybean rust (AmSR) endemic to this region, was found in Adjuntas naturally infecting numerous wild and cultivated legumes, particularly Lablab purpureus (3). Symptoms of AmSR in L. purpureus appeared as reddish-brown spots on the underside of the leaves with three to four uredia per lesion. On February 12, 2011, leaf samples of soybean in beginning pod-fill (R5) and beginning-maturity (R8) growth stages were collected in a winter nursery at EEI and found to have small brown specks with chlorotic haloes on the underside of the leaves and leaf sections from symptomatic areas indicated an abundance of uredinia. Under the light microscope, urediniospores observed at 40× were morphologically similar to Phakopsora spp. Total DNA was extracted from leaf discs using the Qiagen DNeasy Plant Mini Kit following the methods of Frederick et al. (2). Detection of ASR pathogen was achieved via PCR amplification with Ppa1 and Ppa2 primers that are specific for P. pachyrhizi Syd. After sequencing the amplicon, BLAST analysis of the internal transcribed spacer (ITS) region of the ribosomal RNA genes indicated 100% identity with known P. pachyrhizi sequences in GenBank. The sequence of isolate P. pachyrhizi EEI-2011 was submitted to GenBank as JX994293. No amplification was observed after PCR with species-specific primers Pme1 and Pme2 specific for P. meibomiae (Arthur) Arthur. L. purpureus collected from EEA and Utuado only appears to be infected by P. meibomiae and no mixed infections with P. pachyrhizi were apparent, based on the PCR test. Leaf samples from EEI were sent to the UF Plant Diagnostic Center in Gainesville, FL, where quantitative PCR with primers Ppa1 and Ppa2 confirmed the presence of P. pachyrhizi; while P. meibomiae was not detected with primers Pme1 and Pme2. Pathogenicity tests were conducted on the soybean cv. Williams with isolate EEI-2011. Fifteen-day-old soybean plants were inoculated by attaching an infected and sporulating 1 cm2 piece of soybean leaf from EEI-2011 with an average of 4.5 × 105 urediniospores per cm2 (1). Inoculated plants were placed in a growth chamber at 20°C night and 28°C day temperatures, 80% humidity, and a 12-h light photoperiod. Small reddish brown spots with chlorotic haloes developed 4 to 6 days after inoculation and tan lesions appeared 10 to 15 days later. Mature tan lesions developed in 2 weeks with an average of 2.4 uredinia/lesion. Urediniospores were observed with light microscope and these were morphologically similar to those spores observed in the original diseased samples. Another PCR test confirmed P. pachyrhizi after amplification with the species-specific primers. The pathogenicity test was repeated twice with the same cultivar. To our knowledge, this is the first report of ASR in Puerto Rico and this finding will have implications as another overwintering site for Asian soybean rust in the Caribbean region.
References: (1) C. Estévez de Jensen et al. J. Agric. Univ. P.R. 93:125, 2009. (2) R. D. Frederick et al. Phytopathology 92:217, 2002. (3) B. Vega and C. Estévez de Jensen. J. Agric. Univ. P.R. 94:211, 2010.