Comparison of Mineral Oil, Insecticidal, and Biopesticide Spraying Regimes for Reducing Spread of Three Potato virus Y Strains in Potato Crops.

In-field management of Potato virus Y (PVY) faces challenges caused by the changing availability and environmental acceptability of chemical agents to control aphid vectors of the virus and by proliferation of PVY strains with different symptoms and rates of spread. From 2018 to 2020, foliar spray treatments were compared in field experiments in New Brunswick, Canada, to measure effectiveness at reducing spread of PVYO, PVYN:O, and PVYNTN strains. Mineral oil, insecticide, combined oil and insecticide spray, and a biopesticide (i.e., LifeGard WG) were compared. Insecticide-only and mineral oil-only treatments were not effective, but several combined oil and insecticide treatments and biopesticide treatments significantly reduced PVY spread. The biopesticide was proportionately more effective with recombinant PVYN:O and PVYNTN strains, possibly by exciting the plant's hypersensitive resistance response, caused naturally only in cultivar 'Goldrush' by PVYO. Pesticide residue analysis showed that mineral oil increased the retention of pyrethroid insecticide in the potato foliage longer than with insecticide applied alone, which may explain the beneficial synergistic effect of combined sprays for reducing PVY spread. Tuber yields were generally unchanged in chemical insecticide treatments but were slightly lower in biopesticide treatment. The cost per PVY treatment was competitive across all effective treatments, including biopesticide; however, there was some revenue loss from lower yield with the biopesticide. This biopesticide is certified organic, however, and thus a small premium on the price for organic production could offset this yield deficit.

Proliferation of Recombinant PVY Strains in Two Potato-Producing Regions of Canada, and Symptom Expression in 30 Important Potato Varieties with Different PVY Strains.

Potato virus Y (PVY) exists as several strains with distinct symptomology and tuber yield effects in different potato varieties. Recently, new recombinant strains have proliferated and dominated local populations around the world. In this study, PVYO, PVYN:O, PVYN-Wi, and PVYNTN strains were tracked across Canada from 2014 to 2017, showing rapid evolution of populations away from the traditionally dominant PVYO to recombinants PVYN-Wi (western Canada) and PVYNTN (eastern Canada). Simultaneously, 30 potato varieties were inoculated with PVYO, PVYN:O, and PVYNTN in controlled greenhouse experiments. Foliar symptoms of primary (mechanical inoculation mimicking aphid infection) and secondary (tuber seedborne) infection were cataloged, and tuber yield measured. On average, and generally similar in primary and secondary infection, symptom expression and yield reduction were most severe with PVYO, followed by PVYN:O and PVYNTN. Strong mosaic symptoms were most commonly expressed with PVYO infection, and only seen with PVYN:O or PVYNTN in 15 and 3 varieties, respectively. Across variety-strain combinations, yield reduction was correlated with symptom severity, most strongly in PVYO-infected plants (e.g., AC Chaleur, Beljade, Envol, Norland, and Pacific Russet), and four varieties exhibited tuber necrotic ringspot disease with PVYNTN (AC Chaleur, Envol, Pacific Russet, and Yukon Gold).

A Genetic Survey of Pyrethroid Insecticide Resistance in Aphids in New Brunswick, Canada, with Particular Emphasis on Aphids as Vectors of Potato virus Y.

Aphids are viral vectors in potatoes, most importantly of Potato virus Y (PVY), and insecticides are frequently used to reduce viral spread during the crop season. Aphids collected from the potato belt of New Brunswick, Canada, in 2015 and 2016 were surveyed for known and novel mutations in the Na-channel (para) gene, coding for the target of synthetic pyrethroid insecticides. Specific genetic mutations known to confer resistance (kdr and skdr) were found in great abundance in Myzus persicae (Sulzer) (Hemiptera: Aphididae), which rose from 76% in 2015 to 96% in 2016. Aphids other than M. persicae showed lower frequency of resistance. In 2015, 3% of individuals contained the resistance mutation skdr, rising to 13% in 2016 (of 45 species). Several novel resistance mutations or mutations not before reported in aphids were identified in this gene target. One of these mutations, I936V, is known to confer pyrethroid resistance in another unrelated insect, and three others occur immediately adjacent and prompt similar chemical shifts in the primary protein structure, to previously characterized mutations associated with pyrethroid resistance. Most novel mutations were found in species other than M. persicae or others currently tracked individually by the provincial aphid monitoring program, which were determined by cytochrome C oxidase I (cox1) sequencing. Through our cox1 DNA barcoding survey, at least 45 species of aphids were discovered in NB potato fields in 2015 and 2016, many of which are known carriers of PVY.

Strawberry vein banding virus isolates in eastern Canada are molecularly divergent from other isolates.

The complete sequence of a strawberry vein banding virus (SVBV) isolate collected in Nova Scotia, Canada, and designated NS8, was determined. The 7,856-nucleotide circular double-stranded DNA genome contains seven open-reading frames (ORFs), which is consistent with other SVBV isolates and other members of the genus Caulimovirus. Comparison of NS8 with other whole-genome sequences retrieved from databases revealed that NS8 shares the highest sequence similarity (96.5% identity) with isolate China (accession number HE681085) and the lowest (88.3% identity) with clone pSVBV-E3 (accession number X97304). Despite the overall high sequence similarity between NS8 and China, the coat protein encoding ORF IV of NS8 shares only 90.9% sequence identity with the China isolate. Phylogenetic analysis at the complete-genome level placed NS8 and all Chinese isolates in one clade and clone pSVBV-E3 in a separate clade. Interestingly, phylogenetic analysis of all available ORF IV sequences, including those retrieved from databases and newly sequenced samples in this study from Canada, revealed three distinct clades. All Canadian isolates grouped together as one clade, pSVBV-E3 and several others from Europe, Egypt and the USA grouped as a second clade, and isolates from China formed a third clade. These results demonstrate that SVBV is more divergent than previously reported.

RT-PCR and real-time RT-PCR methods for the detection of potato virus Y in potato leaves and tubers.

Potato virus Y (PVY) is a major threat to potato crops around the world. It is an RNA virus of the family Potyviridae, exhibiting many different strains that cause a range of symptoms in potato. ELISA detection of viral proteins has traditionally been used to quantify virus incidence in a crop or seed lot. ELISA, however, cannot reliably detect the virus directly in dormant tubers, requiring several weeks of sprouting tubers to produce detectable levels of virus. Nor can ELISA fully discriminate between the wide range of strains of the virus. Several techniques for directly detecting the viral RNA have been developed which allow rapid detection of PVY in leaf or tuber tissue, and that can be used to easily distinguish between different strains of the virus. Described in this chapter are several protocols for the extraction of RNA from leaf and tuber tissues, and three detection methods based upon reverse-transcription-PCR (RT-PCR). First described is a traditional two-step protocol with separate reverse transcription of viral RNA into cDNA, then PCR to amplify the viral cDNA fragment. Second described is a one-step RT-PCR protocol combining the cDNA production and PCR in one tube and one step, which greatly reduces material and labor costs for PVY detection. The third protocol is a real-time RT-PCR procedure which not only saves on labor but also allows for more precise quantification of PVY titre. The three protocols are described in detail, and accompanied with a discussion of their relative advantages, costs, and possibilities for cost-saving modifications. While these techniques have primarily been developed for large-scale screening of many samples for determining viral incidence in commercial fields or seed lots, they are also amenable to use in smaller-scale research applications.

Effects of Crop Management Practices on Current-Season Spread of Potato virus Y.

The current-season spread of Potato virus Y (PVY) was monitored in 19 fields under various management practices in New Brunswick, Canada, through the 2011 and 2012 growing seasons. The focus of this study was to evaluate the role of seedborne PVY inoculum, aphid vector abundance, and the numbers, timing, and types of insecticide and mineral oil sprays, and to confirm the reliability and forecasting capacity of midseason PVY testing. In each field, 100 to 110 virus-free plants were identified shortly after emergence and were assessed four times from early July to early September (after top-kill) with enzyme-linked immunosorbent assay (ELISA) and reverse-transcription polymerase chain reaction (RT-PCR) to track PVY spread. In addition, tubers harvested during development in August and after top-kill were grown-out in the greenhouse for ELISA testing. PVY spread to selected virus-free plants varied widely, ranging from 0 to 76.2% across all studied fields. Of the 19 fields over two seasons, 10 fields were planted with no detectable seedborne PVY, and they showed 0 to 8.7% (mean 2.9%) PVY spread by harvest. The remaining nine study fields with 0.9 to 5.8% seedborne PVY showed 1 to 76.2% (mean 15.2%) PVY spread by harvest. PVY spread was detected in most fields during midseason testing with ELISA and RT-PCR; all tests correlated well with final PVY rates after top-kill, though RT-PCR detection in developing tubers was most sensitive and correlated. Logistic regression modeling was used to identify major factors in PVY spread, including seedborne PVY, early-season aphid abundance, and the numbers of insecticide and mineral oil sprays. The best-fitting model, constructed using these factors as well as a measurement of July PVY incidence (ELISAJuly), strongly explained PVY spread by harvest, with the most significant management factor being the number of mineral oil sprays supplemented with insecticide used during the growing season. A similar model fitted without the ELISAJuly did not adequately predict ultimate PVY spread. The analysis suggests that mineral oil alone was effective at lowering PVY spread, and more effective when combined with insecticide, particularly when used early in the season. No evidence was found for differences in PVY spread across the eight cultivars used or across the range of mineral oil application rates, whereas some evidence was found for differences in the effectiveness of different insecticide types.

Circadian photosynthetic reductant flow in the dinoflagellate Lingulodinium is limited by carbon availability.

Circadian rhythms are the observed outputs of endogenous daily clocks and are thought to provide a selective advantage to cells adapted to daily light/dark cycles. However, the biochemical links between the clock and the overt rhythms in cell physiology are generally not known. Here, we examine the circadian rhythm in O2 evolution by cultures of the dinoflagellate Lingulodinium, a rhythm previously ascribed to rhythmic electron flow through photosystem II. We find that O2 evolution rates increase when CO2 concentrations are increased, either following addition of DIC or a rapid decrease in culture pH. In medium containing only nitrate as an electron acceptor, O2 evolution rates mirror the circadian rhythm of nitrate reductase activity in the cells. Furthermore, competition between photosynthetic electron flow to carbon and to nitrate varies in its relative efficiency through the day-night cycle. We also find, using simultaneous and continuous monitoring of pH and O2 evolution rates over several days, that while culture pH is normally rhythmic, circadian changes in rates of O2 evolution depend not on the external pH but on levels of internal electron acceptors. We propose that the photosynthetic electron transport rhythm in Lingulodinium is driven by the availability of a reductant sink.

An external delta-carbonic anhydrase in a free-living marine dinoflagellate may circumvent diffusion-limited carbon acquisition.

The oceans globally constitute an important sink for carbon dioxide (CO(2)) due to phytoplankton photosynthesis. However, the marine environment imposes serious restraints to carbon fixation. First, the equilibrium between CO(2) and bicarbonate (HCO(3)(-)) is pH dependent, and, in normal, slightly alkaline seawater, [CO(2)] is typically low (approximately 10 mum). Second, the rate of CO(2) diffusion in seawater is slow, so, for any cells unable to take up bicarbonate efficiently, photosynthesis could become carbon limited due to depletion of CO(2) from their immediate vicinity. This may be especially problematic for those dinoflagellates using a form II Rubisco because this form is less oxygen tolerant than the usually found form I enzyme. We have identified a carbonic anhydrase (CA) from the free-living marine dinoflagellate Lingulodinium polyedrum that appears to play a role in carbon acquisition. This CA shares 60% sequence identity with delta-class CAs, isoforms so far found only in marine algae. Immunoelectron microscopy indicates that this enzyme is associated exclusively with the plasma membrane. Furthermore, this enzyme appears to be exposed to the external medium as determined by whole-cell CA assays and vectorial labeling of cell surface proteins with (125)I. The fixation of (14)CO(2) is strongly pH dependent, suggesting preferential uptake of CO(2) rather than HCO(3)(-), and photosynthetic rates decrease in the presence of 1 mm acetazolamide, a non-membrane-permeable CA inhibitor. This constitutes the first CA identified in the dinoflagellates, and, taken together, our results suggest that this enzyme may help to increase CO(2) availability at the cell surface.

Dynamics of fluxes through photosynthetic complexes in response to changing light and inorganic carbon acclimation in Synechococcus elongatus.

Cyanobacteria acclimate to environmental inorganic carbon (C(i)) concentrations through re-organisations of photosynthetic function and the induction of carbon concentrating mechanisms (CCMs), which alter and constrain their subsequent acclimation to changing light. We grew cells acclimated to high C(i) (4 mM) or low C(i) (0.02 mM), shifted them from 50 micromol m(-2) s(-1) to 500 micromol m(-2) s(-1), and quantified their photosynthetic performance in parallel with quantitation of allocations to key indicator macromolecules. Pigments cell(-1) declined, PsbA (PS II), AtpB (ATP Synthase), RbcL (Rubisco) and GlnA (Glutamine Synthetase) increased, and PsaC (PS I) remained stable through the light shift. The increase in these protein pools was slower and smaller in low C(i) cells, but acted in both cell types to re-normalise the electron fluxes through the catalytic complexes back toward values before the light shift (for PsbA and GlnA) or even below the initial flux per complex (for RbcL). In contrast, an increased electron flux per PsaC was sustained for at least 6 h after the increase in light. Initially, high levels of PS II cell(-1) and PS II connectivity in high C(i) cells caused a more rapid net photoinactivation of PS II in high C(i) cells than in low C(i) cells, depressing the rate of PS II-specific electron transport (PS II ETR) to levels similar to linear ETR (net O(2) evolution minus respiration). In low C(i) cells, PS II ETR remained in excess of linear ETR and may have helped maintain CCM activity. The pool sizes of PsbA, AtpB and GlnA correlated with cellular growth rate, and changed at similar rates in high C(i) and low C(i) cells when expressed on a generational rather than chronological timescale, which has implications for differing ecology of high and low C(i) cells under variable natural light.

Large reallocations of carbon, nitrogen, and photosynthetic reductant among phycobilisomes, photosystems, and Rubisco during light acclimation in Synechococcus elongatus strain PCC7942 are constrained in cells under low environmental inorganic carbon.

Synechococcus elongatus strain PCC7942 cells were grown in high or low environmental concentrations of inorganic C (high-C(i), low-C(i)) and subjected to a light shift from 50 micromol m(-2) s(-1) to 500 micromol m(-2) s(-1). We quantified photosynthetic reductant (O(2) evolution) and molar cellular contents of phycobilisomes, PSII, PSI, and ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) through the light shift. Upon the increase in light, small initial relative decreases in phycobilisomes per cell resulted from near cessation of phycobilisome synthesis and their dilution into daughter cells. Thus, allocation of reductant to phycobilisome synthesis dropped fivefold from pre- to post-light shift. The decrease in phycobilisome synthesis liberated enough material and reductant to allow a doubling of Rubisco and up to a sixfold increase in PSII complexes per cell. Low-C(i) cells had smaller initial phycobilisome pools and upon increased light; their reallocation of reductant from phycobilisome synthesis may have limited the rate and extent of light acclimation, compared to high-C(i) cells. Acclimation to increased light involved large reallocations of C, N, and reductant among different components of the photosynthetic apparatus, but total allocation to the apparatus was fairly stable at ca. 50% of cellular N, and drew 25-50% of reductant from photosynthesis.

Carbon status constrains light acclimation in the cyanobacterium Synechococcus elongatus.

Acclimation to one environmental factor may constrain acclimation to another. Synechococcus elongatus (sp. PCC7942), growing under continuous light in high inorganic carbon (Ci; approximately 4 mm) and low-Ci (approximately 0.02 mm) media, achieve similar photosynthetic and growth rates under continuous low or high light. During acclimation from low to high light, however, high-Ci cells exploit the light increase by accelerating their growth rate, while low-Ci cells maintain the prelight shift growth rate for many hours, despite increased photosynthesis under the higher light. Under increased light, high-Ci cells reorganize their photosynthetic apparatus by shrinking the PSII pool and increasing Rubisco pool size, thus decreasing the photosynthetic source-to-sink ratio. Low-Ci cells also decrease their reductant source-to-sink ratio to a similar level as the high-Ci cells, but do so only by increasing their Rubisco pool. Low-Ci cells thus invest more photosynthetic reductant into maintaining their larger photosystem pool and increasing their Rubisco pool at the expense of population growth than do high-Ci cells. In nature, light varies widely over minutes to hours and is ultimately limited by daylength. Photosynthetic acclimation in S. elongatus occurs in both high and low Ci, but low-Ci cells require more time to achieve acclimation. Cells that can tolerate low Ci do so at the expense of slower photosynthetic acclimation. Such differences in rates of acclimation relative to rates of change in environmental parameters are important for predicting community productivity under variable environments.

Changes in macromolecular allocation in nondividing algal symbionts allow for photosynthetic acclimation in the lichen Lobaria pulmonaria.

• The lichen Lobaria pulmonaria survives large seasonal environmental changes through physiological acclimation to ambient conditions. • We quantitated algal cell population, cell division and key macromolecular levels associated with photosynthesis and nitrogen metabolism in L. pulmonaria sampled from four seasons with contrasting environmental conditions in a deciduous forest. • The algal symbiont population did not vary seasonally and cell division was restricted to the newest thallus margins. Nevertheless the symbiont concentrations of chlorophyll, PsbS, PsbA, and RbcL changed significantly through the seasons in the nondividing algal cells from older thallus regions. • L. pulmonaria reversibly allocated resources toward photochemical electron generation and carbohydrate production through the spring, summer and fall, and towards photoprotective dissipation in the cold, high-light winter. Our study shows that large seasonal molecular acclimation in L. pulmonaria occurs within a nearly stable, nondividing algal cell population that maintains photosynthetic capacity through many years of changing environmental cues.