Two New Bacterial Pathogens of Peanut Causing Early Seedling Decline Disease Identified in the Texas Panhandle

Peanut (Arachis hypogaea L.) is cultivated in tropical and subtropical regions of the world as an important source of oil and protein. Until now, bacterial wilt, caused by Ralstonia solanacearum, was the only known bacterial disease of peanut. In 2020, widespread incidence of poor stand establishment were observed in multiple production fields planted to the Spanish-type peanut varieties in the Texas Panhandle. The observed symptoms included seed rot, pre- and post-emergence damping-off, poor seedling vigor and death, and poorly developed root systems with little or no nodule formation. Subsequent diagnosis of symptomatic seedlings recovered two bacterial species identified by BLAST using 676 bp and 661 bp 16S rRNA fragments as a R. species and a Pantoea sp., respectively. To investigate a possible causative role of these bacteria in the observed peanut disease, the pathogenicity of the two isolates was evaluated under greenhouse conditions relying on Koch’s postulates. Cell suspensions of the two bacteria, separately and in combination, were used to inoculate seeds of a Valencia-type peanut variety with no history of the disease and found to be pathogenic on the resultant seedling plants. Symptoms that developed on the inoculated plants were similar to the symptoms initially observed in the field, including seed rot, pre- and post-emergence damping off, poor seedling vigor and root establishment. The two bacteria were also successfully recovered from inoculated and symptomatic plants, thus satisfying Koch’s postulates. Given the early onset of symptom development on affected seeds and seedlings, a seedborne origin of the disease, described here as early-decline bacterial disease of peanut, was investigated in the same batches of peanut seeds that were planted, as well as seeds later harvested in some of the affected fields. Identical bacterial species, on the basis of 16S rRNA identity, were recovered from all of the seeds evaluated indicating that the bacteria are both seedborne and seed-transmissible. Multi-locus sequence analysis (MLSA) involving six genes (dnaK, fumC, gyrB, murG, trpB, and tuf) showed that these new strains are most closely related to R. pickettii and P. dispersa, but also phylogenetically distinct. The two bacteria were designated Ralstonia sp. strain B265 and Pantoea sp. strain B270. Losses from the disease in affected fields in 2020 averaged fifty percent ($1.12 million) from a total of nine production fields. Findings from this study provide evidence for two new bacterial pathogens of peanuts capable of infecting Spanish and Valencia peanut varieties.

First report of Neopestalotiopsis protearum causing seed rot on Camellia oleifera in China

Camellia oleifera, an evergreen small tree or shrub with high medicinal and ecological values, is mainly distributed in subtropical regions of China. Camellia oil obtained from Camellia oleifera seeds is rich in unsaturated fatty acids and unique flavors, and has become a rising high-quality edible vegetable oil in south of China (Zhuang 2008). The tea-oil tree Camellia oleifera plays important economic and ecological roles in Hunan province. During collecting trips, seeds of C. oleifera with disease symptoms have been observed in almost all oil-tea forests. In lab, the seeds can be infected by wounds and directly, however, wound infection is more rapid. In oil-tea forests, the wound of seed is often caused by external factors such as mechanical and insects. Symptomatic seeds exhibited brown rot symptoms with irregular, black spots, brown necrosis of the kernels, and accounted for 65% of the surveyed seeds (Fig. 1). Rotted seeds were surface-sterilized for 1 min in 75% ethanol, 3 min in 1% sodium hypochlorite, then rinsed for 2 min in sterile water and blotted on dry sterile filter paper. Discolored seed tissues were cut into pieces of 3 mm × 3 mm using a sterile scalpel, placed on potato dextrose agar (PDA) medium, and then incubated for 7 days at 25°C with a 12-h photoperiod. After 7 days of incubation, circular fungal colonies with dense aerial mycelium, produced black, wet spore masses. Four-septate conidia were ellipsoidal to obovoid, measuring 24 (22 to 26) × 6.5 (6 to 7) µm (n = 30). Conidia had three median cells, which were dark brown, with a single basal hyaline appendage, 4 (3.5 to 4.5) µm long, and two to four (usually three) apical hyaline appendages, 32 (27 to 35) µm long, similar to these recorded by Crous et al. (2011). Two single-spore isolates cultured on PDA medium were selected for DNA extraction. The ITS region was amplified using primers ITS5 and ITS4 (White et al. 1990). The partial translation elongation factor 1-alpha (tef1-α) gene region was amplified using primers EF1-728F (O'Donnell et al. 1998) and EF-2 (Carbone & Kohn 1999). The partial β-tubulin (tub2) was amplified using primers T1 and Bt2b (Glass & Donaldson 1995). The sequences of ITS (MW391815), tef1-α (MW398222), and tub2 (MW398223) were submitted to GenBank. BLAST analysis demonstrated that these sequences were 99%~100% similar to the sequences of ITS (MH553959), tef1-α (MH554377), and tub2 (MH554618) published for Neopestalotiopsis protearum. Phylogenetic analysis revealed that all the representative isolates recovered from symptomatic Camellia oleifera seeds showed 91% bootstrap support with Neopestalotiopsis protearum isolate in references (Fig. 2). Pathogenicity tests were conducted on 20 healthy seeds. We wounded the seeds by a sterilized needle on the middle position, and put the 5-mm-diameter agar plugs with actively grown mycelia (strain HNWC04) or pure PDA on the wound. We then covered the wounds with clean masking tape to prevent contamination and desiccation. After inoculation, the seeds were kept at 90 to 100% relative humidity at 25°C in a greenhouse for 3 weeks and monitored daily for lesion development. Twenty days after inoculation, all the seeds inoculated presented similar typical symptoms observed under natural conditions, whereas the control seeds showed no symptoms. Koch’s postulates were fulfilled by reisolating the same fungus and verifying its colony and morphological characters as Neopestalotiopsis protearum. To our knowledge, this is the first report of Neopestalotiopsis protearum causing oil-tea seed rot in China.

Trichoconiella padwickii (stackburn disease).

T. padwickii, previously known as Alternaria padwickii, is an asexually reproducing fungus that infects seeds of rice [Oryza sativa]. It is one of several fungi responsible for seed discolouration, seed rot and seedling blight, but has also been detected as a sheath-rotting pathogen (Naeimi et al., 2003). It occurs in southern Asia and in countries on other continents worldwide, but its presence in mainland North America is not confirmed. Transport to and transmission in new areas may be prevented by use of tested clean seed. Where the pathogen is already present, application of seed treatments should reduce disease incidence, but the fungus has an undetermined ability to survive as sclerotia in plant debris and soil.

Identification of Quantitative Disease Resistance Loci Toward Four Pythium Species in Soybean

In this study, four recombinant inbred line (RIL) soybean populations were screened for their response to infection by Pythium sylvaticum, Pythium irregulare, Pythium oopapillum, and Pythium torulosum. The parents, PI 424237A, PI 424237B, PI 408097, and PI 408029, had higher levels of resistance to these species in a preliminary screening and were crossed with “Williams,” a susceptible cultivar. A modified seed rot assay was used to evaluate RIL populations for their response to specific Pythium species selected for a particular population based on preliminary screenings. Over 2500 single-nucleotide polymorphism (SNP) markers were used to construct chromosomal maps to identify regions associated with resistance to Pythium species. Several minor and large effect quantitative disease resistance loci (QDRL) were identified including one large effect QDRL on chromosome 8 in the population of PI 408097 × Williams. It was identified by two different disease reaction traits in P. sylvaticum, P. irregulare, and P. torulosum. Another large effect QDRL was identified on chromosome 6 in the population of PI 408029 × Williams, and conferred resistance to P. sylvaticum and P. irregulare. These large effect QDRL will contribute toward the development of improved soybean cultivars with higher levels of resistance to these common soil-borne pathogens.

Morphological and molecular characterization of Fusarium graminearum Schwabe as a causal agent of Hyssopus officinalis L. seed rot

Symptoms of seed rot of Hyssopus officinalis L. were noticed during seed health testing in 2018. According to morphological and cultural characteristics, isolates belong to Fusarium spp. and Alternaria spp.. Based on morphological and pathogenic properties, as well as sequence analysis, isolate designated as 4003/3 was identified as Fusarium graminearum deposited in NCBI gene bank under Acc. Number MK061542. To our knowledge F. graminearum as the causal agent of Hyssopus officinalis L. seed rot in Serbia was noticed for the first time.

First report of Pythium ultimum causing damping-off of Sugar beet (Beta vulgaris. L) in Montana, USA

Sugar beet (Beta vulgaris L.) is a globally important crop for sugar. In May 2019, sugar beet seedlings were observed with wilting, lodging and a few were dead in Glendive (46.970170, -104.838204), Montana. Symptoms appeared near the soil line as the stem (hypocotyl) turned dark brown to black with characteristic thread-like infections which resembled Pythium damping-off. It affected approximately 10% of the growing seedlings. Diseased sugar beet root tissues were excised with a sterile scalpel and small pieces (10 mm²) were surface sterilized with 70 % ethanol for 30 seconds, rinsed twice with autoclaved water, air-dried and transferred to potato dextrose agar (PDA) media amended with pimaricin-vancomycin-PCNB (Conway, 1985). Four plates were incubated at 25° C in the dark (Masago et al., 1977) and two weeks later white, dense colony was observed (Zhang et al., 2018). The terminal smooth, globose oogonia (average 18.5 µm in diameter) and antheridia (average 14.5 × 9.5 µm) extended below the oogonium were observed via VWR N. A. 0.30 microscope. The morphological features of the four isolates were consistent with Pythium ultimum Trow (Watanabe, 2002). Genomic DNAs (NORGEN BIOTEK CORP, Fungi DNA Isolation Kit #26200) of four isolates were used for polymerase chain reaction (PCR) with the ITS6-ITS7 primers (Taheri et al., 2017). Subsequently, PCR products were flushed by E.Z.N.A ®Cycle Pure Kit, OMEGA and four samples were sent for Sanger sequencing to GenScript (GenScript, Piscataway, NJ). The sequences were identical and submitted to GenBank, NCBI (accession no. MN398593). The NCBI Blast analysis showed 100% sequence homology to Pythium ultimum with the following GenBank accessions; KF181451.1, KF181449.1 and AY598657.2. Pathogenicity test was done on sugar beet with the same isolates in the greenhouse. Two week old, pythium culture was mixed with vermiculite and perlite mixer (PRO-MIX FLX) in the plastic trays (24´´ x 15´´× 3˝), (22 °C, 75% Relaive Humidity). Sterile water (500 ml/each tray) was added in the mixer to provide sufficient moisture. Twenty seeds of cv. Hilleshog 4302 were sown in the tray, and the trays were replicated thrice with inoculated and mock treatments. Plants were watered as needed to maintain adequate soil moisture conducive for plant growth and disease development. Seven days after sowing, 50% and 100% germination was observed in the inoculated and control treatments, respectively. At the beginning of the second week, 30% post-emergence damping-off was observed in the inoculated treatments. Diseased seedlings were gently pulled out from the pots where similar symptoms were observed in the sugar beet seedlings as described previously. No incidence of disease was observed in mock-treated seedlings. Consistent reisolation of Pythium ultimum was morphologically and molecularly confirmed from the diseased seedlings, thus fulfilling Koch’s postulates. Pythium spp identification is prerequisite to develop effective management of pre and post-emergence damping-off. Pythium ultimum was previously reported in Nebraska to cause sugar beet seed rot and pre-emergence damping-off (Harvenson 2006). To our knowledge, this is the first report of Pythium ultimum causing damping-off on sugar beet in the Sidney factory district in Montana.