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Ascochyta blight (AB) of pulse crops (chickpea, field pea, and lentils) causes yield loss in Montana, where 1.2 million acres was planted to pulses in 2016. Pyraclostrobin and azoxystrobin, quinone outside inhibitor (QoI) fungicides, have been the choice of farmers for the management of AB in pulses. However, a G143A mutation in the cytochrome b gene has been reported to confer resistance to QoI fungicides. A total of 990 isolates of AB-causing fungi were isolated and screened for QoI resistance. Out of these, 10% were isolated from chickpea, 81% were isolated from field peas, and 9% isolated from lentil. These were from a survey of grower’s fields and seed lots (chickpea = 17, field pea = 131, and lentil = 21) from 23 counties in Montana sent to the Regional Pulse Crop Diagnostic Laboratory, Bozeman, MT, United States for testing. Fungicide-resistant Didymella rabiei isolates were found in one chickpea seed lot each sent from Daniels, McCone and Valley Counties, MT, from seed produced in 2015 and 2016. Multiple alignment analysis of amino acid sequences showed a missense mutation that replaced the codon for amino acid 143 from GGT to GCT, introducing an amino acid change from glycine to alanine (G143A), which is reported to be associated with QoI resistance. Under greenhouse conditions, disease severity was significantly higher on pyraclostrobin-treated chickpea plants inoculated with QoI-resistant isolates of D. rabiei than sensitive isolates (p-value = 0.001). This indicates that where resistant isolates are located, fungicide failures may be observed in the field. D. rabiei-specific polymerase chain reaction primer sets and hydrolysis probes were developed to efficiently discriminate QoI- sensitive and - resistant isolates.

Didymella pisi is the primary causal pathogen of Ascochyta blight (AB) of dry pea in Montana. Diagnosis of AB is challenging because there are six different species that cause AB worldwide and that can co-occur. Additionally, agar plate identification of D. pisi is challenging due to its slow growth rate. Currently, there are no PCR-based assays developed for specific detection of D. pisi or any fungal pathogen in the AB complex of dry pea. In this study, we evaluated simple sequence repeat (SSR) primer pairs for their specificity and sensitivity in real-time and conventional SSR-PCR both in vitro and in planta. The specificity of the assay was determined by testing DNA of 10 dry pea varieties, fungal species in the AB complex, and fungal species associated with dry pea. To avoid false-negative results, plant and fungal DNA markers were included as controls in a conventional multiplex SSR-PCR, to amplify any plant or fungal DNA in the absence of the D. pisi SSR target. SYBR Green SSR-quantitative PCR (qPCR) detection was conducted using the same primer pairs but in a uniplex format. D. pisi was specifically amplified, whereas other fungi and host DNA were not. Also, sensitivity experiments showed that the detection limit was 0.01 ng of DNA of D. pisi for both assays and 100 conidia in SSR-qPCR. These assays are valuable diagnostic tools for the detection of D. pisi.

Maize (Zea mays L.) is a major food and fodder crop cultivated on 1.54 million ha in the Democratic Republic of the Congo (DRC). In December 2013, unusually severe chlorotic mottle symptoms and pale green streaks were observed in local varieties (Mudishi 1 and 2, Bambou, Kasayi, H614, H613, and Mugamba) and exotic varieties (H520, H624, H403, HDK8031, and ZM607) in Beni, Lubero, and Rutshuru territories at 1,015 to 1,748 m elevation in North Kivu Province. Symptoms were prominent on newly emerging leaves that later developed marginal necrosis resembling the symptoms of maize lethal necrosis (MLN), caused by a dual infection of Maize chlorotic mottle virus (MCMV, genus Machlomovirus) and Sugarcane mosaic virus (SCMV, genus Potyvirus). Each of these viruses, but particularly MCMV, is also known to cause severe mosaic and mottling symptoms in maize (4). In January 2014, symptomatic and asymptomatic samples (n = 20) from disease-affected fields in Beni and Lubero provinces were collected for virus testing using Whatman FTA Classic Cards (1) and analyzed for MCMV (2681F: 5'-ATGAGAGCAGTTGGGGAATGCG and 3226R: 5'-CGAATCTACACACACACACTCCAGC) and SCMV (8679F: 5'-GCAATGTCGAAGAAAATGCG and 9595R: 5'-GTCTCTCACCAAGAGACTCGCAGC) by reverse transcription (RT)-PCR (4). Samples were also analyzed for Maize streak virus (MSV, genus Mastrevirus), an endemic virus in DRC, by PCR using MSV specific primers (MSV215-234: CCAAAKDTCAGCTCCTCCG and MSV1770-1792: TTGGVCCGMVGATGTASAG) (3). A DNA product of expected size (~520 bp) resulted only for MCMV in all the symptomatic plant samples. None of the samples tested positive for SCMV or MSV. RT-PCR analyses were performed to ascertain the absence of potyviruses using the degenerate potyvirus primers (CIFor: 5'GGIVVIGTIGGIWSIGGIAARTCIAC and CIRev: 5'ACICCRTTYTCDATDATRTTIGTIGC3') (2) were also negative. Occurrence of MCMV in symptomatic samples was further confirmed by antigen-coated plate (ACP)-ELISA using anti-MCMV rabbit polyclonal antibodies produced at the Virology Unit, IITA, Ibadan, Nigeria. The RT-PCR product of MCMV was purified and sequenced in both directions (GenBank Accession No. KJ699379). Pairwise comparison of 518 bp nucleotide sequence corresponding to p32 and p37 open reading frames of MCMV by BLASTn search revealed 99.8% nucleotide sequence identity with an MCMV isolate from Kenya (JX286709), 98 to 99% identity with the isolates from China (JQ982468 and KF010583), and 96% identity with the isolates from the United States (X14736 and EU358605). MCMV is a newly emerging virus in Africa, first detected during a severe MLND outbreak in 2011 in Kenya (4). This disease has since become a serious threat to maize production in East Africa. MCMV has been reported in maize from Kenya, Rwanda, Tanzania, and Uganda. To our knowledge, this is the first report of MCMV occurrence in DRC. This finding confirms the further geographic expansion of MCMV and illustrates the need for further studies to identify vectors and also create awareness about the disease and to strengthen surveillance to prevent its further spread in the continent. References: (1) O. J. Alabi et al. J. Virol. Met. 154:111, 2008. (2) C. Ha et al. Arch. Virol. 153:25, 2008. (3) K. E. Palmer and E. P. Rybicki. Arch. Virol. 146:1089, 2001. (4) A. Wangai et al. Plant Dis. 96:1582, 2012.