Castanea Sativa Research Articles

Uticaj različitih podloga na porast micelije gljive Cryphonectria parasitica (Murrill) M.E. Barr

It is a well-known fact that a large number of parasitic and saprophytic fungi grow on sweet chestnut trees. However, the greatest damage is caused by C . parasitica which causes "sweet chestnut blight" and leads to its dieback. Hosts other than Castanea species include the following families: Aceraceae, Betulaceae, Fagaceae, Anacardiaceae, Juglandaceae i Magnoliaceae, where it grows as a saprophyte. The exception, according to some authors, is oak, where it can also occur as a parasite. Mycelial growth of C. parasitica was tested on media made of the bark of sweet chestnut (Castanea sativa), manna ash (Fraxinus ornus), sycamore maple (Acer pseudoplatanus), sessile oak (Quercus petraea), common yew (Taxus baccata), hazel (Corylus avellana), small-leaved linden (Tilia cordata), Norway maple (Acer platanoides) and English walnut (Juglans regia). After 28 days, the medium was not completely overgrown only in the variants with sycamore maple and Norway maple bark added to the medium.

Biological Richness in the Chestnut (Castanea Sativa) Forests at the Western of the Cantabrian Range

Biological Richness in the Chestnut (Castanea Sativa) Forests at the Western of the Cantabrian Range

The Use of qPCR to Detect Cryphonectria parasitica in Plants.

Cryphonectria parasitica is a fungal pathogen that causes lethal bark necrosis in chestnut. A duplex qPCR allowing detection of the pathogen and its host, Castanea sativa, is described. The method can be used for early detection of the pathogen in chestnut bark tissues with an internal control of false-negative results caused by PCR inhibitors and/or DNA extraction failure. A positive amplification control of qPCR that allows detection of any deviation from a normal qPCR run based on a control chart is also described. As C. parasitica is a regulated pathogen in Europe, the protocol also provides information on the way to collect and handle bark samples to fulfil biosecurity rules.

Metabolites derived from fungi and bacteria suppress in vitro growth of Gnomoniopsis smithogilvyi, a major threat to the global chestnut industry

IntroductionChestnut rot caused by the fungus Gnomoniopsis smithogilvyi is a disease present in the world’s major chestnut growing regions. The disease is considered a significant threat to the global production of nuts from the sweet chestnut (Castanea sativa). Conventional fungicides provide some control, but little is known about the potential of biological control agents (BCAs) as alternatives to manage the disease.ObjectiveEvaluate whether formulated BCAs and their secreted metabolites inhibit the in vitro growth of G. smithogilvyi.MethodsThe antifungal potential of BCAs was assessed against the pathogen through an inverted plate assay for volatile compounds (VOCs), a diffusion assay for non-volatile compounds (nVOCs) and in dual culture. Methanolic extracts of nVOCs from the solid medium were further evaluated for their effect on conidia germination and were screened through an LC–MS-based approach for antifungal metabolites.ResultsIsolates of Trichoderma spp., derived from the BCAs, significantly suppressed the pathogen through the production of VOCs and nVOCs. The BCA from which Bacillus subtilis was isolated was more effective in growth inhibition through the production of nVOCs. The LC–MS based metabolomics on the nVOCs derived from the BCAs showed the presence of several antifungal compounds.ConclusionThe results show that G. smithogilvyi can be effectively controlled by the BCAs tested and that their use may provide a more ecological alternative for managing chestnut rot. The in vitro analysis should now be expanded to the field to assess the effectiveness of these alternatives for chestnut rot management.

Genetic diversity of Cryphonectria parasitica causing chestnut blight in South Tyrol (northern Italy)

European chestnut (Castanea sativa) is threatened by the invasive fungus Cryphonectria parasitica, which causes chestnut blight. The virulence of the fungus can be reduced by a group of mycoviruses that can spread among vegetatively compatible strains through hyphal anastomosis. Hypovirulent isolates are used as biocontrol agents, but their efficiency can be diminished by restricted hyphal anastomosis if the variability of vegetative compatibility (vc) types in a population is high. Sexual reproduction could increase the vc type diversity and further complicate biocontrol in a region. Therefore, knowledge of genetic diversity of C. parasitica is important to assess the effectiveness of a biological control program. The present study was performed in the Autonomous Province of Bozen-Bolzano (South Tyrol) in northern Italy, where chestnut cultivation provides an additional income to farmers. The genetic characterization of C. parasitica isolates from 35 chestnut stands and one forest population in different districts of South Tyrol was performed based on the analysis of vegetative incompatibility loci, the mating type locus, and the internal transcribed spacer (ITS) region. In this study, a total of 23 different vc types were found all over South Tyrol with a Shannon diversity index of 1.86. EU-2, EU-1, and EU-13 were the most widespread vc types comprising 51%, 13%, and 9% of the fungal isolates, respectively. Both mating types were present in the region with a ratio close to 1:1. Three different haplotypes were identified based on ITS sequence analysis, which pointed to two introduction events of the fungus to the region and allowed placing C. parasitica from South Tyrol into a larger phylogeographic context.

Models of Silviculture for Portuguese Species: Old and New Guidelines

Models of silviculture are used for the selection and scheduling of the silvicultural practices from installation to the end of the production cycle. Most of the published models of silviculture are for even aged stands. Those for mixed and uneven aged stands are scarce. The goal of this study was the development of models of silviculture for Portuguese species; with examples for Pinus pinea, Quercus suber Quercus rotundifolia, Pinus pinaster and Castanea sativa. Five stand structures were considered (pure even aged, mixed even aged, temporary mixed even aged, pure uneven aged and mixed uneven aged). For mixed stands were considered two species admixtures and for uneven aged 2, 3 and 4 cohorts. The local level models of silviculture developed in this study are conceptual frameworks where guidelines can be derived to define the model of silviculture for a stand, including target stand structure and production cycle, according to management goals, species and site. Overall, it was intended to stress the importance of defining models of silviculture that are flexible to accommodate a wide range of management options and that should be dynamic in space and time. These models cannot be used directly but have to be adapted to each stand.

Susceptibility levels of some chestnut cultivars and genotypes to the chestnut gall wasp in Turkey

The Asian chestnut gall wasp (ACGW) is one of the most important pests threatening most Castanea species. The best management strategy against this pest can be establishing new orchards with resistant cultivars along with biocontrol (parasitoid Torymus sinensis Kamijo (Hymenoptera: Torymidae)). In Turkey, ACGW was first detected in 2014 at Gac?k village of Yalova province. Bursa, ?stanbul, Sakarya, Kocaeli, Bal?kesir, Bilecik, D?zce, Giresun, Bart?n, Zonguldak, Sinop and ?zmir provinces are also infected with ACGW. This study was carried out to determine the levels of susceptibility of chestnut cultivars/genotypes to ACGW. The study was carried out in Yalova province between 2016 and 2018. In total, 15 cultivars/genotypes were involved in the study, including European (C. sativa) chestnuts (Albayrak, Alt?nay, ?Erfelek?, ?Osmano?lu?, Sal?pazar?, ?Serdar?, ??nal?), interspecific hybrids (?Marigoule? and BDB-L) and complex hybrids (?Aky?z?, ?Macit 55?, ?Ali Nihat?, A9, A55, A56) were tested in the study. In April 2016, five- to seven-year-old seedlings were ?bark? grafted, and susceptibility to the ACGW was evaluated by recording the ratio of infected buds (%), visual assessment of the damage (severity), and calculations of an infestation index. Among the cultivars and genotypes tested in the study, the ?Aky?z? cultivar was determined to exhibit the highest resistance against the ACGW. To fully understand the resistance mechanism of the ?Aky?z? cultivar, molecular and biochemical studies should be done and compared with other results.

Fruit characteristics, defoliation, forest floor and soil properties of sweet chestnut (Castanea sativa Mill.) forests in İstanbul-Turkey

The defoliation and fruit characteristics of natural Castanea sativa Mill. forests were annually monitored between 2014 and 2016 in İstanbul, Turkey. The soil and forest floor properties were also investigated and evaluated according to the stand development stages. Comparisons were made with ANOVA and Tukey HSD tests; the relationship between fruit yield and the properties of the soil and forest floor were tested with correlation analysis; and allometric regression models were developed for fruit yield with DBH (diameter at breast height) and (DBH)2H. The total mass was 509-652 g/m2, N mass was 7.67-9.70 g/m2 and C mass was between 165.75 g/m2 and 183.28 g/m2 in the forest floor in the development stages. The soil texture was loam-clay loam, soil C concentration was between 0.3% and 1.92%, N concentration was 0.08-0.32%, the EC was very low (33-84 μS/cm), and the pH was acidic (5 pH). The properties of the forest floor and soil were not significantly different from the development stages. The defoliation rates increased significantly every year in each development stage. The fruit yield was between 183.51 kg/ha and 298.27 kg/ha, and fruit was not detected in the smallest development stage (SDF). The fruit yields were quite low in comparison with other natural C. sativa forests. However, in each year in the study period, fruit yields were negatively correlated with mass and C and N content and positively correlated with N concentration in the H layer of the forest floor. There was not a significant difference in fruit yield over the years, and it had a low relation with DBH and (DBH)2H (R2 = 0.34 and R2 = 0.23, respectively). The fruits? characteristics significantly fluctuated over the years. As a result, low fruit yield and low relationships with properties of the forest floor and soil might be attributed to the former coppice management and possible health problems.

Melisopalynological potential of chestnut (Castanea sativa Mill.) in Bosnia and Herzegovina

Tame chestnut (Castanea sativa Mill.) is a woody plant species from the family Fagaceae. Chestnut belongs to the group of honey-bearing plants with a high production of pollen and nectar, and honey is characterized by specific chemical and organoleptic properties and a high content of minerals. Its biogeographical distribution is limited to only three areas on the territory of Bosnia and Herzegovina. The aim of the work is to determine the distribution and representation of the pollen of this honey plant in honey samples originating from Bosnia and Herzegovina. For research purposes, 100 honey samples were collected directly from the producer. Based on the micromorphological characteristics of the pollen grains, honey plants, and the exact number of their pollen grains were identified. A total of 4520 chestnut pollen grains were identified in 40 analyzed melissopalynological profiles of honey. It was concluded, that the specific geographical and ecological distribution of chestnuts in the territory of Bosnia and Herzegovina directly affects the melissopalynological composition of the samples, as well as the botanical characteristics of the honey.

Ecological structure: production of organic impregnation material from mussel shell and combustion

In the research, sea mussel shell (Chamelea gallina) powders were impregnated on the samples of Eastern spruce (Picea orientalis (L.) Link.) and Anatolian chestnut (Castanea sativa Mill.) by dipping method at different concentrations (1%, 5%, 10%, 15%). To investigate the level of use in the wood industry and especially its effects against fire; adhesion, thermogravimetric analysis (TGA), limiting oxygen index (LOI) test measurements were carried out. According to the TGA results, while the residue quantity in the spruce wood sample was the highest at 5%, the residue amount in the chestnut wood sample was the highest at 15%. With increasing amounts of mussel shell powder, the limiting oxygen index values in both wood species samples increased. As a result, it was discovered that impregnating wood samples with mussel shell powder improved the wood's fire resistance.

HIGHLIGHTING THE MELISSOPALYNOLOGICAL AND PHYSICOCHEMICAL CHARACTERISTICS OF AYDER-RİZE (TURKEY)

The aim of this research is characterizing the honey produced in Ayder/Rize-Turkey. In this context 20 honey samples were collected from Ayder in 2018. The melissopalynological and physicochemical analysis of the honey samples were done by this research. According to the melissopalynological analysis 40 taxa belong to the 22 plant families were identified as botanical sources of the investigated honey samples. Nine of the investigated samples (sample no 1-9) were evaluated as monofloral and the others as multifloral honeys. As a result of melissopalynological analysis, while pollen belong to the Castanea sativa taxon were found in dominant ratios in some of the investigated samples, the pollen belongs to the Castanea sativa, Trifolium repens, Lotus corniculatus, Coronilla orientalis taxa were found as secondary in some other honey samples. Total pollen number in 10 gram honey (TPN 10) values of the samples were found between 7 732 and 167 147 by melissopalynological analysis. The first step of the physicochemical analsyis was the moisture analysis and the values found between 15,8% and 18,8% (mean value:7,01±0,98 %). The fructose/glucose analysis were done by High Performance Liquid Chromatography (HPLC) and the values for fructose found as: 26,43-35,57g/100g (mean: 31,39±2,41 g/100g), glucose values as; 20,11-30,58 g/100g (mean: 26,83±2,16 g/100g). Also fructose/glucose ratio was found as 1,03-1,34 (mean: 1,17±0,10). Hydroxymethylfurfural (HMF) and total phenolic acids analysis were done also by HPLC. The results for HMF analysis is between 0,7- 11,31 ppm (mean: 3,8±2,6 ppm ), for total phenolic acids the value are found between 88,92±0,04 mgGAE/kg - 196,17±0,10 mgGAE/kg (mean: 121,98±0,1 mgGAE/kg). As a step of chemical analysis; the volatile compounds of the honey samples were determined by Gas Chromatography and Mass Spectrometry (GC-MS). The compounds belong to the aldehydes, aliphatic acids and esters, alcohols, hydrocarbons, carboxylic acids and their esters, ketones, terpenes, fatty acids and their esters groups were found as a result of GC-MS analysis. The compounds belong to the carboxylic acids and their esters, fatty acids and their esters identified in higher ratios compare to the other compounds. Since, there is little detailed published information about the quality and properties of Ayder-Rize honey, the current study aims to characterize the honey belong to the this region.

Linking landscape structure and vegetation productivity with nut consumption by the Cantabrian brown bear during hyperphagia

In bears, reproduction is dependent on the body reserves accumulated during hyperphagia. The Cantabrian brown bear mainly feeds on nuts during the hyperphagia period. Understanding how landscape heterogeneity and vegetation productivity in human-dominated landscapes influence the feeding habits of bears may therefore be important for disentangling species-habitat relationships of conservation interest. We determined the spatial patterns of nut consumption by brown bears during the hyperphagia period in relation to landscape structure, characteristics of fruit-producing patches and vegetation productivity. For this purpose, we constructed foraging models based on nut consumption data (obtained by scat analysis), by combining vegetation productivity data, topographical variables and landscape metrics to identify nut foraging patterns during this critical period for bears. The average wooded area of patches where scats were collected and where the nuts that the bears had consumed were produced was larger than that of the corresponding patches where nuts were not produced. For scats collected outside of nut-producing patches, the distance between the scats and the patches was greatest for chestnut-producing patches. Elevation, Gross Primary Production (GPP) and the Aggregation Index (AI) were good predictors of acorn consumption in the models. Good model fits were not obtained for data on chestnut consumption in bears. The findings confirm that brown bears feeding on nuts show a preference for relatively large, highly aggregated patches with a high degree of diversity in the landscape pattern, which may help the bears to remain undetected. The nut prediction model highlights areas of particular importance for brown bears during hyperphagia. The human presence associated with sweet chestnut forest stands or orchards may make bears feel more vulnerable when feeding.

Development and Optimization of a Topical Formulation with Castanea sativa Shells Extract Based on the Concept “Quality by Design”

The proposed study aims to develop and optimize a topical formulation with Castanea sativa shells extract considering the concept of Quality by Design, focusing on a planned development that consider the vulnerabilities of the entire process through risk analysis tools and design of experiments (DoE). A Box–Behnken design with three factors and three levels was used as a statistical tool for the execution of the DoE and the analysis of the response surface methodology responses. The independent variables studied were the quantity of sodium lauryl sulfate (%) (X1), beeswax (%) (X2) and macadamia oil (%) (X3); the dependent variables were pH (Y1), viscosity (Y2) and adhesiveness (Y3). According to the mathematical model, the optimal formulation contains 0.93% of sodium lauryl sulfate, 5.00% of beeswax and 10.00% of macadamia oil. The optimal formulation with the extract was prepared and characterized over the time, regarding organoleptic and technological characteristics, allowing conclusions to be reached regarding its stability. The formulation presented a pleasant odor and was light brown in color, it also demonstrated pseudoplastic-thixotropic behavior and a small reduction in the formulation consistency after 30 days of storage. This study demonstrated the efficiency of the Quality by Design methodology to understand the product variability, supporting that this approach favors a better understanding of the whole process and enables to design a robust development stage, reducing costs and generating high-quality products.

The effect of the conversion of chestnut (Castanea sativa Mill.) forests to orchards on soil fertility and nutrient content in leaves

Chestnut (Castanea sativa Mill.) is a valuable tree species of high ecological importance, which could constitute a key development of the mountainous economy. The method of ecosystem management causes significant changes in soil properties, which in turn could lead to changes in the availability of soil resources. In Greece, in 1979, the use of the right for exploitation of chestnut clusters, located in public forests was granted in exchange for a price, for the purpose of the grafting of the chestnut trees for nut production. The aim of the present study was to investigate the effects of land use change from old forest sites to chestnut cultivation for nut production on soil fertility and nutrition. The research was carried out in the area of Griva, a village located on Mount Paiko, in northern Greece. For the needs of the research, 10 sampling plots were installed. The results showed higher soil organic N and exchangeable Ca and K accumulation in the Castanea orchards, compared to the forest sites. The foliar P, K and Fe contents in the forest sites were higher compared to the orchards. In contrast, the foliar Ca concentrations were higher in the orchards, compared to the forest sites. After 40–45 years from land use change, the no-tillage management system of the Castanea orchards, with plant residues’ recycling and herbaceous vegetation cover and the use of the necessary fertilization, conserved the soil fertility adequate, strengthening the sustainable development of ecosystems.

Hydrothermal and high-pressure processing of chestnuts - Dependence on the storage conditions

High-pressure processing (HPP) is an emergent technology for nonthermal pasteurization of foods, which here demonstrates the potential to be used by the chestnut industry. This study evaluated HPP as a post-harvest processing technology to control molds and insect larvae proliferation in chestnuts (Castanea sativa Mill.) and improve the fruit's shelf-life. Chestnuts were processed by HPP (400, 500, and 600 MPa for 5.0 min at 20 °C) and the conventional hydrothermal technology (HT, 50 °C for 45 min). Untreated chestnuts were also used as a control group. Shelf-life studies were conducted for 60 d at i) 25 °C and 40 % relative humidity (RH); and ii) 5 °C and 70 % RH. After the treatment and over the storage period, parameters such as color, texture, weight loss, nutritional composition, ascorbic acid, and fungal load were evaluated. The results showed that immediately after the HPP treatments, the chestnuts became slightly softer than HT samples. Fruit treated by HPP maintained their nutritional composition and ascorbic acid content. During the first 30 d of storage, no changes of color were observed in treated fruit. Also, the shelf-life of chestnuts treated at 600 MPa was extended by 40 d, compared to HT, where no molds at a detectable level and insect larvae were observed when stored at 5 °C. This storage condition also reduced the fruit weight losses. Compared with HT, HPP has short processing times, which is an important advantage. In addition, the observed changes of texture as softening and easy peeling can be useful for obtaining ready-to-eat products since chestnuts looked partially cooked. However, its possible application at the industrial level should be adequately investigated based on the promising results obtained.

GIS-Based Assessment of the Chestnut Expansion Potential: A Case-Study on the Marvão Productive Area, Portugal

Sweet chestnut is a relevant species in Europe for the production of timber and fruit, alongside environmental effects such as biodiversity of protection against soil erosion. In Portugal, chestnut is cultivated mainly for fruit production, in two areas, in the North and the South of the country, with moderate water deficit and low slope and at altitudes higher than 500 m. The current area (845 ha) of the southern so-called Marvão Protected Designation of Origin, of a fortyfold lower order of magnitude by comparison with the Northern productive area, has a significant expansion potential, given its similarity with contiguous areas in the same region. In this context, the main objective of the present work was the evaluation through geographic information analysis of that expansive potential, by comparison of physiographic profiling of the current production area with contiguous areas. A GIS-based characterization of current and potential chestnut areas in Marvão is presented. The methodology involved (i) digital profiling of the main classes/values of the geographical spatial ecological fingerprint considering topography, soil and microclimate variables in the areas currently occupied with sweet chestnut stands and (ii) the evaluation of the distribution of that environmental fingerprint in the whole Marvão productive area, for extending the cultivation to contiguous areas with a similar ecological fingerprint. An enlarged 9889 ha chestnut area was proposed, allocated for high forest stands aiming at agroforestry fruit production and coppiced stands for timber production and environmental protection, corresponding to 4590 ha and 5299 ha, respectively. Fruit production was proposed to field slopes of 0–4% and 4–8%, and altitudes between 400 m and 500 m. Presumable high-quality sites allocated to temporary dry/irrigated cultivations were also proposed for fruit production, in the same slope classes and altitudes higher than 500 m. Timber production and environmental protection were proposed for slopes within 8−12% and >12% ranges. This selection took into account the logistical feasibility facilitated in lower slopes for intensive mechanized management operations. This methodology permits a future field evaluation of site indexes, productivity, and correlations between environmental variables and stand biometry.

Anti-inflammatory activities of Italian Chestnut and Eucalyptus honeys on murine RAW 264.7 macrophages

The aim of this study was to evaluate for the first time the anti-inflammatory effects of chestnut (Castanea sativa) and Eucalyptus (Eucalyptus occidentalis) honeys collected in Italy on murine RAW 264.7 macrophages stressed with the bacterial endotoxin lipopolysaccharide (LPS). Pre-treatment with the two honeys markedly decreased apoptosis and stimulated wound healing in macrophages stressed with LPS, where they also reduced the intracellular levels of ROS and nitrite. Additionally, treatment with LPS decreased the levels of glutathione and suppressed the antioxidant enzyme activities, which were significantly (p < 0.05) increased in the presence of the two honeys, through the modulation of Nrf2 expression; in LPS-stressed macrophages, Chestnut or Eucalyptus honeys protected against inflammation by regulating the main inflammatory biomarkers, including TNF-α, IL-10 and iNOS, through the decrease of NF-кB expression. Finally, they improved mitochondrial respiration and the main related parameters.

Anthropogenic and landscape features are associated with ink disease impact in Central Italy

AbstractInk disease of sweet chestnut (Castanea sativa) caused by the oomycetes Phytophthora cinnamomi and P. x cambivora is the limiting factor for chestnut cultivation in several European regions. The objective of this study was to explore how the spatial landscape heterogeneity affects the distribution pattern of ink disease over a large chestnut area in Central Italy using an approach that combined remote sensing, ground truthing activities and GIS. A multivariate model was developed that explained a large proportion of the variance of the impact of the disease using the density of roads and drainage networks as predictor variables. The association of these landscape elements, specifically with ink disease foci, also provides practical tool to improve the accuracy of monitoring of this disease and the preparation of risk maps.

Origin Identification of Hungarian Honey Using Melissopalynology, Physicochemical Analysis, and Near Infrared Spectroscopy

The objective of the study was to check the authenticity of Hungarian honey using physicochemical analysis, near infrared spectroscopy, and melissopalynology. In the study, 87 samples from different botanical origins such as acacia, bastard indigo, rape, sunflower, linden, honeydew, milkweed, and sweet chestnut were collected. The samples were analyzed by physicochemical methods (pH, electrical conductivity, and moisture), melissopalynology (300 pollen grains counted), and near infrared spectroscopy (NIRS:740–1700 nm). During the evaluation of the data PCA-LDA models were built for the classification of different botanical and geographical origins, using the methods separately, and in combination (low-level data fusion). PC number optimization and external validation were applied for all the models. Botanical origin classification models were >90% and >55% accurate in the case of the pollen and NIR methods. Improved results were obtained with the combination of the physicochemical, melissopalynology, and NIRS techniques, which provided >99% and >81% accuracy for botanical and geographical origin classification models, respectively. The combination of these methods could be a promising tool for origin identification of honey.

Honey botanical origin and honey-specific protein pattern: Characterization of some European honeys

Honey adulteration generates low quality products on the market. The study aimed to find a simple, specific and less time-consuming method than standard melissopalynology only, for monitoring honey botanical and geographical origin. In this study 42 honey samples from different sources were examined for their botanical origin by using melissopalynology and their specific protein patterns by electrophoresis on SDS polyacrylamide gels (SDS-PAGE). The melissopalynological analysis consisted of counting all melliferous pollen, non-melliferous pollen and honeydew elements to identify the species of pollen present in each sample. The honey samples had predominant pollen from Apiaceae, Boraginaceae, Brassicaceae, Compositae H, Fabaceae (Trifolium sp.) and Fagaceae family. From the Fagaceae family, the most important species was Castanea sativa Mill., while the Boraginaceae was represented by Echium sp. and Myosotis sp. SDS-PAGE showed that the different origin honeys shared protein bands between 45 and 85 kDa (animal origin proteins, i.e. major royal jelly proteins, enzymes) and that specific proteins (presumably plant origin proteins) can be attributed to individual honey types. This study shows that the combination of melissopalynology and SDS-PAGE is a useful tool for modern discrimination between different kinds of honey, even without performing specific protein identification.