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Montana leads the production of dry pea in the United States. About 530,000 acres were planted to pea in 2019, accounting for 48% of the total national production ( USDA-NASS 2019 ). A predominant foliar disease of dry pea in Montana is Ascochyta blight, which is caused by multiple fungal pathogens including Didymella pisi, Peyronellaea pinodes, and Peyronellaea pinodella. D. pisi is the predominant pathogen causing Ascochyta blight of dry pea in Montana. Recently, an anticipated shift in pathogen composition has been observed in northeastern Montana from D. pisi to P. pinodes. Also, a Phoma sp. was found associated with infected dry pea seeds and included in this study. To characterize these fungi, we evaluated the effects of temperature (15, 20, 25, and 30°C) on mycelial growth rate and sporulation. The optimum temperature for mycelial growth and sporulation was either 20 or 25°C depending on the species. Analysis of variance supported that at all evaluated temperatures, Phoma sp. had the highest growth rate and produced more spores than the other species (P value < 0.001). In pathogenicity assays, P. pinodes caused more severe disease than the other species when inoculated on pea plants (cv. Carousel, P value ≤ 0.001). The Phoma sp. was not pathogenic. Peameal agar (PMA) was developed as a diagnostic tool for these pathogens. On PMA, the fungal species showed different mycelial morphology, which was used to visually discriminate them. Results from this study will be used as a base to understand the adaptability, pathogenicity and aggressiveness, and current status and changes in the population composition of fungal species causing Ascochyta blight of dry pea in Montana and North America.

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.

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 ascochyta blight 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 Ascochyta blight-causing fungi were isolated and screened for QoI resistance. Out of these, 10% were isolated from chickpea, 81% were isolated from dry peas, and 9% isolated from lentil. These were from a survey of grower’s fields and seed lots (chickpea = 17, pea = 131, and lentil = 21) from 23 counties in Montana sent to the Regional Pulse Crop Diagnostic Laboratory, Bozeman, MT 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 revealed 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 reportedly 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). These results suggest that disease control may be inadequate at locations where resistant isolates are present. D. rabiei-specific polymerase chain reaction primer sets and hydrolysis probes were developed to efficiently discriminate QoI-resistant and - sensitive isolates

Soybean production in Tanzania is steadily increasing, driven by growing demand from feed and livestock producers and also for human consumption. Soybean production area has increased from 795 ha in 2003 to 4,100 ha in 2013 (FAO 2014). Major soybean production is in the Morogro, Ruvuma, Iringa, and Mbeya regions. During a soybean rust disease survey conducted in May 2014 in Morogoro in the southern highlands of Tanzania, soybean plants with phyllody and witches’-broom disorder typical of phytoplasma infection was observed on cultivar, Uyole Soya#1 in a farmer’s field at Msufini village (6°17′0.099″ S; 37°28.791″ E). Symptoms consisted of shoot proliferation, reduced size of the leaflets and petiole, proliferation of axillary shoots with shortened internodes, phyllody, and viriscence. About 50% of the plants assessed (n = 20) from one plot in a farmer’s field were infected. Symptomatic and asymptomatic leaves were collected for total genomic DNA extraction and PCR amplification using Candidatus phytoplasma universal primer pair P1 and P7 for 16S-23S ribosomal RNA encoding region (Sharmila et al. 2004). PCR amplicons of expected size (∼1,700 bp) resulted from the templates of the symptomatic samples only. They were directly sequenced in both orientations and the nucleotide sequence was submitted to GenBank (Accession No. KP205526). A BLASTn search revealed that the phytoplasma sequences had a nucleotide sequence identity of 99% with those of 16SrII group phytoplasma associated with phyllody and witches’-broom disease of soybean in Malawi (HQ845208) and Mozambique (HQ840717). Phylogenetic analysis revealed the clustering of these strains with members of 16SrII group. The virtual restriction fragment length polymorphism (RFLP) pattern derived from these sequences using iPhyClassifier software (Zhao et al. 2009) was similar to the reference pattern of the 16SrII subgroup C (cactus phytoplasma, AJ293216), with a pattern similarity coefficient of 0.99. Previous reports of phytoplasma occurrence in Tanzania were related to coconut lethal decline disease caused by 16SrIV-C subgroup phytoplasma (Bila et al. 2014). To our knowledge, this is the first report of the occurrence of 16SrII-C subgroup phytoplasma causing phyllody and witches’-broom disease in soybean in Tanzania. The occurrence of phyllody and witches’-broom disease was first recognized in soybean in Malawi and Mozambique in 2010 (Kumar et al. 2011). Detection of the same pathogen in the diseased soybean plants in Tanzania suggest either spread of 16SrII phytoplasma from neighboring countries or 16SrII phytoplasma may be widespread in asymptomatic wild or weed hosts in southern Africa, spreading to crop hosts like soybean because of intensive cultivation. Nonetheless, this finding underscores the need for better understanding of epidemiology of 16SrII phytoplasma, especially its natural hosts and vectors, to prevent its adverse impacts on soybean production in Eastern and Southern Africa. Further surveys in soybean production areas in Tanzania are necessary to estimate the extent of spread and economic importance

Soybean production in Tanzania is steadily increasing, driven by growing demand from feed and livestock producers and also for human consumption. Soybean production area has increased from 795 ha in 2003 to 4,100 ha in 2013 (FAO 2014). Major soybean production is in the Morogro, Ruvuma, Iringa, and Mbeya regions. During a soybean rust disease survey conducted in May 2014 in Morogoro in the southern highlands of Tanzania, soybean plants with phyllody and witches’-broom disorder typical of phytoplasma infection was observed on cultivar, Uyole Soya#1 in a farmer’s field at Msufini village (6°17′0.099″ S; 37°28.791″ E). Symptoms consisted of shoot proliferation, reduced size of the leaflets and petiole, proliferation of axillary shoots with shortened internodes, phyllody, and viriscence. About 50% of the plants assessed (n = 20) from one plot in a farmer’s field were infected. Symptomatic and asymptomatic leaves were collected for total genomic DNA extraction and PCR amplification using Candidatus phytoplasma universal primer pair P1 and P7 for 16S-23S ribosomal RNA encoding region (Sharmila et al. 2004). PCR amplicons of expected size (∼1,700 bp) resulted from the templates of the symptomatic samples only. They were directly sequenced in both orientations and the nucleotide sequence was submitted to GenBank (Accession No. KP205526). A BLASTn search revealed that the phytoplasma sequences had a nucleotide sequence identity of 99% with those of 16SrII group phytoplasma associated with phyllody and witches’-broom disease of soybean in Malawi (HQ845208) and Mozambique (HQ840717). Phylogenetic analysis revealed the clustering of these strains with members of 16SrII group. The virtual restriction fragment length polymorphism (RFLP) pattern derived from these sequences using iPhyClassifier software (Zhao et al. 2009) was similar to the reference pattern of the 16SrII subgroup C (cactus phytoplasma, AJ293216), with a pattern similarity coefficient of 0.99. Previous reports of phytoplasma occurrence in Tanzania were related to coconut lethal decline disease caused by 16SrIV-C subgroup phytoplasma (Bila et al. 2014). To our knowledge, this is the first report of the occurrence of 16SrII-C subgroup phytoplasma causing phyllody and witches’-broom disease in soybean in Tanzania. The occurrence of phyllody and witches’-broom disease was first recognized in soybean in Malawi and Mozambique in 2010 (Kumar et al. 2011). Detection of the same pathogen in the diseased soybean plants in Tanzania suggest either spread of 16SrII phytoplasma from neighboring countries or 16SrII phytoplasma may be widespread in asymptomatic wild or weed hosts in southern Africa, spreading to crop hosts like soybean because of intensive cultivation. Nonetheless, this finding underscores the need for better understanding of epidemiology of 16SrII phytoplasma, especially its natural hosts and vectors, to prevent its adverse impacts on soybean production in Eastern and Southern Africa. Further surveys in soybean production areas in Tanzania are necessary to estimate the extent of spread and economic importance