Genetic and Pathogenic Characterization of Phacidiopycnis washingtonensis from Apple and Pacific Madrone from the Western United States.

Phacidiopycnis washingtonensis is the cause of speck rot of apple and leaf blight of Pacific madrone in Washington State. In total, 314 isolates were collected from apple production areas in eastern Washington and Pacific madrone in western Washington. Using eight microsatellite markers designed in this study, 58 unique multilocus haplotypes were identified. Only one of the haplotypes was shared between the apple and Pacific madrone populations. Analysis of molecular variance showed no genetic differentiation between the apple and Pacific madrone populations. Genetic variation was present within each subpopulation of apple from different geographic locations. The apple population possessed higher genotypic diversity than the Pacific madrone population, suggesting that isolates from apple may represent an older population and could have been introduced into the native habitat of Pacific madrone. P. washingtonensis likely reproduces asexually because populations examined in this study were not in linkage equilibrium. In pathogenicity tests, representative isolates from apple and Pacific madrone all incited leaf blight on Pacific madrone and speck rot on apple fruit regardless of their host of origin. Overall, our findings indicate that the P. washingtonensis population in Washington State is largely asexual, with high genotypic flow and that apple, crabapple, and Pacific madrone could serve as sources of P. washingtonensis inoculum for these hosts.

Groundwater Quality beneath an Asian Megacity on a Delta: Kolkata's (Calcutta's) Disappearing Arsenic and Present Manganese.

Kolkata, the capital city of West Bengal, exploits groundwater for public water-supply. The groundwater has been reported to be widely polluted by arsenic (As). Analysis for As in 280 groundwaters from across Kolkata, failed to detect As concentrations >10 µg/L from natural processes. Arsenic concentrations between 10 and 79 µg/L found in 14 of the 280 groundwaters are remnant from a pollution-plume emanating from a single factory site where Paris Green, an arsenical pesticide, was manufactured between 1965 and 1985. In 45% of groundwaters sampled, concentrations of Mn exceed 0.4 mg/L, a putative health guideline value for drinking water. Sporadic minor hazards are posed by Pb > 10 µg/L introduced into groundwater from well-fittings, from 4% of groundwaters with F concentrations between 0.75 and 1 mg/L, and the 14% of groundwaters containing more than 500 mg/L Na, concentrations that might contribute to excessive daily intake of Na. Compounding hazards from As, F, Mn, Na, and Pb, shows that 64% of public wells and 40% of municipal wells supply groundwater of suspect quality. Groundwaters comply with WHO Guideline Values for drinking water in terms of Cr, Cu, Co, NO2, NO3, Sb, Se, and U. Aesthetic guideline values for Fe, Mn, SO4, and Cl are exceeded for many groundwaters.

Arsenic in Groundwater: The Deep Late Pleistocene Aquifers of the Western Bengal Basin.

in groundwaters from 145 wells across central West Bengal, India, those from Pleistocene aquifers at depths >70 m beneath paleo-interfluves contain <10 µg/L As. Pleistocene aquifers beneath deep paleo-channels typically host groundwaters containing 10-100 µg/L As at depths between 120 and 180 m. The depth profiles of As and SO4 and the conservative tracers Cl/Br, δ(18)O, and δ(2)H show that the As in Pleistocene groundwater beneath deep paleo-channels is relict and does not arise from migration downward of As-polluted groundwater in overlying aquifers. We postulate that the As was liberated in situ by reduction of minimal iron oxyhydroxides in the gray Pleistocene sands by organic matter infiltrating from riverbeds during late Pleistocene or earliest Holocene times. Mitigation of the widespread As-pollution in shallow aquifers through exploitation of deep Pleistocene aquifers would improve if guided by an understanding of the distribution of buried paleo-channels and paleo-interfluves and the knowledge that As may be present naturally in groundwater at depths >150 m beneath deep paleo-channels.

Palaeosol Control of Arsenic Pollution: The Bengal Basin in West Bengal, India.

Groundwater in the Bengal Basin is badly polluted by arsenic (As) which adversely affects human health. To provide low-As groundwater for As mitigation, it was sought across 235 km(2) of central West Bengal, in the western part of the basin. By drilling 76 boreholes and chemical analysis of 535 water wells, groundwater with <10 µg/L As in shallow aquifers was found under one-third of a study area. The groundwater is in late Pleistocene palaeo-interfluvial aquifers of weathered brown sand that are capped by a palaeosol of red clay. The aquifers form two N-S trending lineaments that are bounded on the east by an As-polluted deep palaeo-channel aquifer and separated by a shallower palaeo-channel aquifer. The depth to the top of the palaeo-interfluvial aquifers is mostly between 35 and 38 m below ground level (mbgl). The palaeo-interfluvial aquifers are overlain by shallow palaeo-channel aquifers of gray sand in which groundwater is usually As-polluted. The palaeosol now protects the palaeo-interfluvial aquifers from downward migration of As-polluted groundwater in overlying shallow palaeo-channel aquifers. The depth to the palaeo-interfluvial aquifers of 35 to 38 mbgl makes the cost of their exploitation affordable to most of the rural poor of West Bengal, who can install a well cheaply to depths up to 60 mbgl. The protection against pollution afforded by the palaeosol means that the palaeo-interfluvial aquifers will provide a long-term source of low-As groundwater to mitigate As pollution of groundwater in the shallower, heavily used, palaeo-channel aquifers. This option for mitigation is cheap to employ and instantly available.

Development of PCR Assays for Diagnosis and Detection of the Pathogens Phacidiopycnis washingtonensis and Sphaeropsis pyriputrescens in Apple Fruit.

Speck rot caused by Phacidiopycnis washingtonensis and Sphaeropsis rot caused by Sphaeropsis pyriputrescens are two recently reported postharvest diseases of apple. Infection by these two pathogens occurs in the orchard but remains latent before harvest. Symptoms develop after harvest and are similar to those of gray mold caused by Botrytis cinerea. Accurate diagnosis of these diseases is important during the fruit inspection process, particularly in the instance of fruit destined for export. Early near-harvest detection of latent infections in apple fruit is an important step to implement relevant pre- and postharvest measures for disease control. The aim of this study was to develop polymerase chain reaction (PCR) assays for diagnosis and early detection of latent infections of apple fruit by P. washingtonensis and S. pyriputrescens. Species-specific primers based on the ribosomal DNA internal transcribed spacer region were designed for use in PCR assays. Conventional and real-time PCR assays were developed and validated using fruit inoculated with P. washingtonensis, S. pyriputrescens, or B. cinerea and compared with identifications using traditional isolation-based assays. For wound-inoculated fruit, the PCR assays consistently provided the correct identification of the pathogen used as the inoculant in 6 h of processing time, compared with 5 to 6 days using culture-based methods. Real-time PCR assays effectively detected latent infections in symptomless stem and calyx tissues of fruit that were inoculated with the pathogens in the orchard during the growing season. The PCR assays provide a rapid, accurate method for diagnosis and early detection of these diseases.

Infection Courts and Timing of Infection of Apple Fruit by Phacidiopycnis washingtonensis in the Orchard in Relation to Speck Rot During Storage.

Phacidiopycnis washingtonensis is the cause of speck rot, a recently reported postharvest fruit rot disease of apple. The pathogen is believed to incite infections in the field, and disease symptoms become evident only during storage. To determine the timing of apple fruit infection in relation to development of speck rot in storage, 'Red Delicious' and 'Fuji' apple fruit were inoculated in the orchard with P. washingtonensis at different times during the growing season, harvested, and monitored for decay development during storage at 0°C. Fruit inoculated in both field and laboratory also were used to identify the infection courts and mode of apple fruit penetration by P. washingtonensis. In all 3 years, stem-end speck rot and calyx-end speck rot developed during cold storage on fruit inoculated during the growing season, regardless of inoculation time; and the incidence of total speck rot in storage increased as the fruit inoculation time approached harvest. On fruit floral parts, the pathogen colonized sepals at higher rates than stamens. Availability of naturally occurring necrotic tissues favored the colonization of the fungus on sepals. Histological studies indicated that infection occurred through micro-cracks on the surfaces of pedicels and sepals of the fruit, and invasion of these tissues was restricted between the cuticle and epidermis. Findings of this study will assist in the development of effective control strategies for speck rot.

First Report of a New Leaf Blight Caused by Phacidiopycnis washingtonensis on Pacific Madrone in Western Washington and Oregon.

In recent years, a leaf blight disease, consisting of browned, desiccated leaves occurring mainly in the lower parts of the canopy, has been observed during wet springs on Pacific madrone (Arbutus menziesii) in western Washington and Oregon. In May 2009 and 2011, severe outbreaks occurred and symptomatic leaves from madrones growing in the region were sampled to determine the causal agent. Two symptoms, leaf necrosis or blotching along the edges and tips of the leaves, and leaf spot, were observed. Small segments of diseased tissue were cut from the leaves, surface-disinfected, rinsed, and plated on malt extract agar. Fifty percent of the leaf blotch and 30% of leaf spot samples yielded a fungus that was fast-growing (20 mm diameter in 4 days at 25°C) and produced colonies that were a pale gray with dark gray reverse and a felty texture. On potato dextrose agar (PDA), pycnidia formed and exuded conidia in peach-colored droplets after 2 weeks under room temperature and light conditions. Pycnidia were spherical and 12.5 to 39.8 µm, average 24.2 µm in diameter. Conidia were hyaline, ovoid, and 5.8 to 8.5 × 3.1 to 4.7 µm (average 7.0 × 3.7 µm). The fungus was identified as Phacidiopycnis washingtonensis based on its morphology (1). To confirm the identity, the internal transcribed spacer (ITS) region of the rDNA was amplified with ITS1/ITS4 primers (2) and sequenced (GenBank Accession Nos. JQ743784 to 86). BLAST analysis showed 100% nucleotide identity with those of P. washingtonensis in GenBank (AY608648). The fungus was also isolated from lesions on green shoots and the petiole and leaf blade of dead attached leaves. To test pathogenicity, 3-year-old Pacific madrone seedlings (three for each isolate) were inoculated with five isolates of the fungus and maintained in the greenhouse (25°C); the experiment was conducted twice. Five leaves from each tree were cold injured (-50°C) at a marked 5 × 5 mm2 area with a commercial aerosol tissue freezing product prior to inoculation and five leaves were not cold injured. A 5-mm-diameter mycelial plug cut from the margin of 6-day-old PDA culture was applied to the marked areas on the upper leaf surface. The inoculated area was covered with moist cheese cloth and wrapped with Parafilm. Leaves treated with blank PDA plugs served as control. Leaves were enclosed in plastic bags to maintain moisture for the first 15 h post inoculation and cheese cloths were removed after 15 days. All cold-injured inoculated leaves showed symptoms of blight starting at 2 weeks after inoculation, and no symptoms appeared on the controls. On non-cold injured inoculated leaves, only one isolate caused symptoms (80% of all leaves). The fungus was re-isolated from diseased leaves. These results suggest that P. washingtonensis is able to cause foliar blight on Pacific madrone when leaves are subjected to cold stress. Increased disease severity on madrone observed in spring 2011 in Washington and Oregon may have been due to predisposition of foliage to extreme cold in November 2010 and February 2011. This fungus has previously been reported to cause a postharvest fruit rot disease on apple fruit and a canker and twig dieback disease of apple and crabapple trees in WA (1). To our knowledge, this is the first report of P. washingtonensis causing a leaf blight disease on Pacific madrone in North America. References: (1) C. L. Xiao et al. Mycologia 97:464, 2005. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

First Report of Sphaeropsis Rot of Apple Caused by Sphaeropsis pyriputrescens in New York.

In March 2012, decayed 'Empire' apple fruit (Malus × domestica Borkh.) were sampled from apples grown in Albion (Orleans County) in New York State and stored in bins for 6 months under controlled atmosphere at a commercial packinghouse. At the packinghouse following storage prior to be packed, the fruit were completely rotten, spongy to firm, and light brown without pycnidia. All fruit rots originated from either stem-end or calyx-end infections but no wound infections were observed. The incidence of fruit with these symptoms in the total decay was relatively low (0.1%). To isolate the causal agent, small fragments of fruit flesh from 12 decayed fruit were cut and placed on potato dextrose agar (PDA) acidified with 0.1% lactic acid. The plates were incubated at 20°C for 4 days and sub-cultured on PDA to obtain a pure culture. The colonies initially appeared with dense hyaline mycelium and later turned light yellow to yellow, and black pycnidia formed after about 2 weeks of incubation under a 24-h fluorescent light at 20°C. Conidia were light brown to brown, clavate to subglobose to irregular, and 15 × 8 µm on average. The fungus was identified as Sphaeropsis pyriputrescens Xiao & J.D. Rogers based on the morphology of the fungus (3). The identity of a representative isolate was further confirmed by analysis of nucleotide sequences of the internal transcribed spacer (ITS) regions amplified using the primers ITS1/ITS4. A BLAST search in GenBank showed that the sequence had 99% homology to an S. pyriputrescens sequence (Accession No. GQ374241). One representative isolate was tested for pathogenicity on apple fruit. Organic 'Red Delicious' apple fruit were surface-disinfected in 0.6% sodium hypochlorite solution for 5 min, rinsed twice with deionized water, and air-dried. Each fruit was wounded with a sterilized finish-nail head (3 mm in depth and 4 mm in diameter) and inoculated by placing a 4-mm-diameter plug from the leading edge of a 4-day-old PDA culture on the wound. Control fruit were treated with sterile PDA plugs. The inoculation site was covered with two layers of moist cheesecloth to avoid dehydration. There were four 10-fruit replicates for each treatment, and fruit were placed in plastic crispers and stored at 4°C for 4 weeks. The experiments were conducted twice. Sphaeropsis rot developed on all inoculated fruit, while no decays appeared on the control fruit. Koch's postulates were fulfilled by reisolating the fungus from the decayed fruit. Sphaeropsis rot is a recently reported postharvest fruit rot disease of apple and pear (1,3). The disease was first observed on 'd'Anjou' pears, and later more serious economic losses were observed in apples in Washington State (1). The disease has also since been reported in British Columbia, Canada (2). To the best of our knowledge, this is the first report of the occurrence of Sphaeropsis rot caused by S. pyriputrescens on apple in New York or in any region outside of the Pacific Northwest in North America. References: (1) Y. K. Kim and C. L. Xiao. Plant Dis. 92:940, 2008. (2) P. L. Sholberg et al. Plant Dis. 93:843, 2009. (3) C. L. Xiao et al. Plant Dis. 88:223, 2004.

Waste-water impacts on groundwater: Cl/Br ratios and implications for arsenic pollution of groundwater in the Bengal Basin and Red River Basin, Vietnam.

Across West Bengal and Bangladesh, concentrations of Cl in much groundwater exceed the natural, upper limit of 10 mg/L. The Cl/Br mass ratios in groundwaters range up to 2500 and scatter along mixing lines between waste-water and dilute groundwater, with many falling near the mean end-member value for waste-water of 1561 at 126 mg/L Cl. Values of Cl/Br exceed the seawater ratio of 288 in uncommon NO(3)-bearing groundwaters, and in those containing measurable amounts of salt-corrected SO(4) (SO(4) corrected for marine salt). The data show that shallow groundwater tapped by tube-wells in the Bengal Basin has been widely contaminated by waste-water derived from pit latrines, septic tanks, and other methods of sanitary disposal, although reducing conditions in the aquifers have removed most evidence of NO(3) additions from these sources, and much evidence of their additions of SO(4). In groundwaters from wells in palaeo-channel settings, end-member modelling shows that >25% of wells yield water that comprises ≥10% of waste-water. In palaeo-interfluvial settings, only wells at the margins of the palaeo-interfluvial sequence contain detectable waste water. Settings are identifiable by well-colour survey, owner information, water composition, and drilling. Values of Cl/Br and faecal coliform counts are both inversely related to concentrations of pollutant As in groundwater, suggesting that waste-water contributions to groundwater in the near-field of septic-tanks and pit-latrines (within 30 m) suppress the mechanism of As-pollution and lessen the prevalence and severity of As pollution. In the far-field of such sources, organic matter in waste-water may increase groundwater pollution by As.

The palaeosol model of arsenic pollution of groundwater tested along a 32 km traverse across West Bengal, India.

The distribution of As-pollution in groundwater of the deltaic aquifers of south-eastern Asia may be controlled by the subsurface distribution of palaeo-channel sediments (As-polluted groundwaters) and palaeo-interfluvial sediments (As-free groundwaters). To test this idea, termed the palaeosol model of As-pollution, we drilled 10 sites, analysed groundwater from 249 shallow wells (screened <107 mbgl), field-tested another 149 for As, and used colour as a guide to the presence or absence of As-pollution in a further 531 wells. Our work was conducted along a 32-km traverse running W to E across southern West Bengal, India. At seven drill sites we logged a palaeo-interfluvial sequence, which occurs as three distinct units that together occupy 20 km of the traverse. These palaeo-interfluvial sequences yield As-free groundwaters from brown sands at depth<100 m. The palaeo-interfluvial sequences are separated by two deep palaeo-channels, which were logged at 3 sites. The palaeo-channel deposits host As-polluted groundwater in grey sands. Our findings confirm the predictions of the palaeosol model of As-pollution. We show again that well-colour can be used both to successfully predict the degree of As-pollution in groundwater, and to locate regions of buried palaeo-interfluve that will yield As-free groundwater for the foreseeable future.

Sedimentological control on Mn, and other trace elements, in groundwater of the Bengal delta.

To reveal what controls the concentration and distribution of possibly hazardous (Mn, U, Se, Cd, Bi, Pb) and nonhazardous (Fe, V, Mo, PO(4)) trace elements in groundwater of the Bengal delta, we mapped their concentrations in shallow groundwater (<60 mbgl) across 102 km(2) of West Bengal. Only Mn is a potential threat to health, with 55% of well water exceeding 0.3 mg/L, the current Indian limit for drinking water in the absence of an alternate source, and 75% exceeding the desirable limit of 0.1 mg/L. Concentrations of V are <3 µg/L. Concentrations of U, Se, Pb, Ni, Bi, and Cd, are below WHO guideline values. The distributions of Fe, Mn, As, V, Mo, U, PO(4), and δ(18)O in groundwater reflect subsurface sedimentology and sources of water. Areas of less negative δ(18)O reveal recharge by sources of evaporated water. Concentrations of Fe, As, Mo, and PO(4) are high in palaeo-channel groundwaters and low in palaeo-interfluvial groundwaters. Concentrations of U, V, and Mn, are low in palaeo-channel groundwaters and high in palaeo-interfluvial groundwaters. Concentrations of Fe and Mn are highest (18 and 6 mg/L respectively) at dual reduction-fronts that form strip interfaces at depth around the edges of palaeo-interfluvial aquifers. The fronts form as focused recharge carries dissolved organic carbon into the aquifer margins, which comprise brown, iron-oxide bearing, sand. At the Mn-reduction front, concentrations of V and Mo reach peak concentrations of 3 µg/L. At the Fe-reduction front, concentrations of PO(4) and As reach concentrations 3 mg/L and 150 µg/L respectively. Many groundwaters contain >10 mg/L of Cl, showing that they are contaminated by Cl of anthropogenic origin and that organic matter from in situ sanitation may contribute to driving reduction.

Serology and immunoglobulin profile in rheumatoid arthritis.

One hundred and twenty cases of clinically diagnosed rheumatoid arthritis, 80 non-rheumatoid cases suffering from various other diseases and 40 healthy individuals were investigated for the presence of rheumatoid factor, quantitation of serum immunoglobulin, demonstration of ANA and LE cell phenomenon. Microlatex agglutination test of serum for rheumatoid factor showed 56.6% positivity in rheumatoid group and 3.7% positivity in non-rheumatoid group. All three serum immunoglobulins (IgG, IgM, IgA) were raised in serum in significant titre in cases of rheumatoid arthritis, whereas only IgA lever was elevated in the group of non-rheumatoid diseases. ANA and LE cell phenomenon were observed in 11.7% and 4.4% cases of rheumatoid arthritis who had severe underlying disease. In non-rheumatoid group, only one of 6 cases of systemic lupus erythematosus showed rheumatoid factor and that too in an insignificant titre (less than 1:20). Synovium and synovial fluid contained plenty of plasma cells and lymphocytes. It has been observed that RF appears first in synovial fluid and it may take several months to a year to attain detectable level in serum.

Spatial aggregation in gravity models: 4. Generalisations and large-scale applications.

"This paper is concerned with applying and extending a methodology for analysing spatial aggregation in gravity models developed in three earlier papers to a larger scale and hence more realistic example of spatial interaction than has been treated so far." Seven models are generated and applied to four levels of aggregation of the spatial interaction pattern in Edmonton, Alberta.

On population growth of cities in a region: a stochastic nonlinear model.

"A stochastic migration model describing the population dynamics in a region is investigated. The model is described by a pair of coupled differential equations with state-dependent stochasticity. Explicit expressions for the time evolution of the moments for the population sizes of cities are obtained from the Fokker-Planck equation. The Stratonovich calculus is employed in the analysis."

Spatial aggregation in gravity models: 2. One-dimensional population density models.

"This paper, the second of four, is concerned with applying a methodology for analysing the spatial aggregation problem in gravity models outlined in the first paper. The methodology is based on a consistent framework for linking measures of pattern in interaction data to the derivation and estimation of related interaction models using spatial information. In this quest, a link is forged between information in data and the parameters of an associated model.... This relationship can be formalised for the case of the continuous one-dimensional interaction model such as the population density model, and this paper is concerned with demonstrating such an application to aggregations of zones in the Reading [England] region."