First Report of Pectobacterium brasiliense causing soft rot on mizuna (Brassica rapa var. japonica) in the United States.

Mizuna (Brassica rapa var. japonica), a member of family Brassicaceae, is a leafy vegetable having phenolic and other compounds beneficial to human health, such as natural antioxidants (Khanam et al. 2012). In October 2020, a field of mizuna (variety: Early) on Oahu island was observed having 20-30% diseased plants. Four randomly selected infected mizuna plants, showing the symptoms of wilt and stem rot (Figure 1A-D), were collected and isolations were made to determine the pathogen. Small sections of infected stems were cut, surface sterilized with 0.6% sodium hypochlorite solution for 30 sec, followed by three consecutive rinses in distilled water. The tissues were macerated in a sterile 1.5 ml centrifuge tube containing 100 µl sterile water-macerated tissues were streaked onto crystal violet pectate medium (CVP) (Hélias et al. 2011) and incubated at 26 ± 2°C for 48 h. Isolated bacterial colonies that formed pits on the CVP plates were re-streaked onto dextrose peptone agar: Peptone (10 g/L), Dextrose (5 g/L) and Agar (17 g/L) (DPA-without tetrazolium chloride; Norman and Alvarez 1989) to obtain purified colonies for DNA isolation using DNeasy Blood and Tissue Kit (Qiagen, Germantown, MA). The two housekeeping genes (dnaA and gapA) were amplified and sequenced following the protocols used by Dobhal et al. (2020) and Boluk et al. (2020), for identity confirmation and phylogenetic analysis. Cleaned PCR products were sent to the GENEWIZ facility (Genewiz, La Jolla, CA) for sequencing of sense and antisense strands. The obtained sequences were aligned, manually edited, and consensus sequences were analyzed with BLASTn using the NCBI GenBank nucleotide and genome databases for identity confirmation. The BLASTn results demonstrated 100% query coverage of all four strains (PL248-PL251); and showed 100% identity of PL248 and PL249, and 99% identity of PL250 and PL251 with Pectobacterium brasiliense. All the sequences were submitted to the NCBI GenBank database under the following accession numbers: dnaA gene MW560271 - MW560274 (PL248 - PL251); and gapA gene MW560275 - MW560278 (PL248 - PL251). Pathogenicity was assessed by artificially inoculating 100 µl bacterial suspension of each strain (PL248 - 1.12x 108 CFU/ml; PL249 - 1.32x 108 CFU/ml; PL 250 - 1.2x 108 CFU/ml and PL251 - 1.15x 108 CFU/ml) onto four-week-old mizuna (variety: Leafy Asian Greens) plants in three replicates, using sterile pipette tips, which was stabbed into stem halfway and wrapped with parafilm. The inoculated plants were well maintained under controlled greenhouse conditions. As negative controls, three plants were inoculated with 100 µl distilled water. Soft rot and wilt symptoms (Figure 1E-H) were observed 24 hours post inoculation. No symptoms were observed on control plants (Figure 1F). All four strains were re-isolated from the inoculated plants and confirmed as P. brasiliense based on resequencing of the dnaA region and 100% homology with the sequences of original strain. In the phylogenetic tree (Figure 2), based on two housekeeping genes (dnaA and gapA), the bacterial strains from mizuna grouped with other P. brasiliense retrieved from the NCBI GenBank database. To our knowledge, this is the first report of P. brasiliense infecting mizuna plants in Hawaii or in the USA and is important because this species is one of the most aggressive pectolytic pathogens in the genus Pectobacterium. Understanding the diversity of different pectolytic phytopathogens is essential to formulating risk mitigation strategies as P. brasiliense could potentially pose a threat to additional vegetable crops, especially the crucifers vegetables (Arizala et al. 2019; Klair et al, 2021).

Nutrient concentrations within and below root zones from applied chicken manure in selected Hawaiian soils.

The objective of this study was to evaluate the effects of chicken manure (CM) application rates on nutrient concentrations within and below the root zone of sweet corn (Zea mays L. subsp. mays) under Hawaiian conditions. The research was conducted in leeward (Poamoho) and windward (Waimanalo) areas of Oahu, Hawaii, where contrasts exist in both climatic and soil conditions. Suction cup were used to collect soil solutions from 30 and 60 cm depths. Soil solutions were collected six times during the growing season at each location and analyzed for different nutrients (N, P, K, Ca, Mg, Na, Fe, Mn, Zn, and Cu), nitrate-nitrogen (NO(3)-N), ammonium-nitrogen (NH(4)-N), electrical conductivity (EC), and pH. Analysis showed that CM rates significantly affected the concentration of macro-nutrients below the root zone at Poamoho and within the root zone at Waimanalo. In general, nutrient concentration increased with the increasing rates of CM application. There was a significant effect of CM on micro-nutrients except below the root zone at Poamoho. CM significantly affected NO(3)-N concentration within the root zone for 15, 60 days after planting (DAP) at Poamoho, and 16, 28 DAP at Waimanalo. The effect was also significant on total nitrogen (N) concentration in the root zone across the two growing seasons at Waimanalo. There was a highly significant correlation between total N and NO(3)-N, and EC within and below the root zone.

Biomass and nutrient concentration of sweet corn roots and shoots under organic amendments application.

Two field experiments were conducted at the Waimanalo research station on the island of O'ahu, Hawaii to study the effect of chicken (CM) and dairy (DM) manures on biomass and nutrient concentration in sweet corn roots and shoots. Sweet corn (super sweet 10, Zea Mays L. subsp. mays) was grown for two consecutive growing seasons under four rates of application (0, 168, 337, and 672 kg ha(-1) total N equivalent) and one time (OTA) or two time (TTA) applications of organic manure types and rates. There were significant effects of types, rates, and number of manure applications on dry biomass and macro- and micro-nutrient concentration in roots and shoots tissues. Results of root tissue indicated a significant accumulation of N and C under CM and DM treatments compared with the control treatment. Manure application rates significantly increased the accumulation of N and C in root tissue. Dry weight of roots and shoots and both macro- and micro-nutrient contents in the plant tissues significantly increased under TTA treatment compared with OTA treatment. There was a significant correlation (r(2) = 0.46 to 0.81) between root biomass, macro-, and micro-nutrient contents during both growing seasons. The results of the study indicates that amending soils with CM at the highest application rate provided the best crop performance in terms of root and shoot biomass, crop N, C, and other macro- and micro-nutrients.