Potato Losses Research Articles

Global gene expression in the common scab pathogen, Streptomyces scabies, exposed to potato microtubers

AbstractStreptomyces scabies causes yield losses in potato by inducing common scab (CS) symptoms on tubers. Little information is available on global gene expression responses in CS‐causing streptomycetes upon interaction with host plants. In this study, expression of 8746 genes was analysed in the type strain of S. scabies (ATCC49173) using a custom‐designed, gene‐specific oligonucleotide microarray. Bacteria were grown in liquid yeast malt extract (YME) medium and 480 genes were found to be up‐regulated 24 h after supplementing the cultures with potato microtubers. Quantitative PCR analysis of eight putative or known virulence genes was consistent with the microarray data. Elevated expression of the thaxtomin synthesis gene txtA and txtR genes regulating synthesis of thaxomin A, the main pathogenicity factor of S. scabies, and production of thaxtomin A were detected in cultures containing microtubers. Unexpectedly, thaxtomin was also detected in cultures lacking microtubers, which indicated that growth in YME induces thaxtomin production in S. scabies. Many but not all microtubers incubated in the cultures of S. scabies for 48 h displayed symptoms, indicating that the microtubers were not equally susceptible even though they were produced under uniform in vitro growth conditions. These factors may have limited the differences in gene expression observed between S. scabies exposed to microtubers compared to controls. This study provides some of the first results on the transcriptome of S. scabies, a widely distributed and important potato pathogen, upon exposure to young developing potato tubers. The findings will inform future studies on interactions of CS‐inducing streptomycetes with potato and means to control the disease.

Effect of gamma irradiation on the physico-mechanical and chemical properties of potato (Solanum tuberosum L.), cv. ‘Kufri Sindhuri’, in non-refrigerated storage conditions

The effects of γ-irradiation doses, 0.04, 0.08, 0.12 and 1kGy, applied at two different postharvest times (5 and 30 days after harvest), were studied on the textural behaviour (puncture force, shear force, work done to puncture and shear, cohesiveness and gumminess), microstructure, reducing sugar, total sugar and tuber losses of potato (Solanum tuberosum L.), cv. ‘Kufri Sindhuri’, during storage at 22°C (RH: 85–90%). The lowest dose (0.04kGy) was sufficient to inhibit sprouting in potatoes exposed on day 5 but not in the tubers exposed on day 30. The irradiated, non-sprouted potatoes maintained their appearance during storage. Potatoes irradiated early appeared more sensitive to radiation-induced damage, resulting in excessive loss of tubers at 1kGy but low doses (up to 0.12kGy) did not increase the susceptibility of the tubers to rotting. No significant differences between reducing sugar and total sugar contents of the control and low dose irradiated tubers were observed after 120d. High dose (1kGy) induced blackening of the bud tissue, increased rotting percentage and poor textural quality. Increasing low doses (up to 0.12kGy) progressively reduced the textural deterioration in the tubers during storage. The scanning electron micrographs of potatoes irradiated with 0.08–0.12kGy showed intact cells with rigid cell walls, accounting for the higher textural values registered by the samples. Among the two treatment timings, ‘K. Sindhuri’ irradiated early after harvest (i.e., on day 5) with 0.08–0.12kGy doses retained higher textural parameters compared to those irradiated after a delay (day 30). The study showed the potential effect of γ-irradiation for enhancing the storage life of potatoes in non-refrigerated storage.

Rapid detection and quantification of viable potato cyst nematodes using qPCR in combination with propidium monoazide

Potato cyst nematodes (PCN), Globodera pallida and Globodera rostochiensis, are obligate parasites of solanaceous plants, causing severe losses in several potato growing areas throughout the world. To date, management of PCN is related to nematode population densities estimated as eggs per gram of soil, without considering the actual number of viable juveniles within the cysts. In classical nematology, the standard method to determine PCN viability is based on a staining assay, using Meldola's blue dye (MB) followed by microscopic visualization of MB‐treated nematodes. Although MB is considered to be reliable in staining embryonated juveniles within eggs and cysts, it is a time‐ and labour‐consuming assay. In the present work, a real‐time PCR (qPCR)‐based method combined with propidium monoazide (PMA), a photoreactive DNA‐intercalating dye, was developed for the quantification of viable PCN. This dye renders exposed DNA of dead cells unable to be amplified by PCR, and thus only DNA from viable/intact PCN juveniles is amplified and detected. The novelty of the present method lies in the simultaneous quantitative and qualitative estimation of viable PCN inocula using species‐specific primers and TaqMan probes. The PMA–qPCR viability method (v‐PCR) developed for the two Globodera species successfully discriminated dead from living specimens in heat‐treated samples and eggs in old and newly formed cysts. Interestingly, the detection of DNA from 34‐year‐old nematode cysts stored at room temperature was observed. In conclusion, the proposed v‐PCR method should prove to be very useful for the routine determination of PCN viability from field samples.

Storage System of Potato in Bangladesh

The study was undertaken to investigate the storage system and seasonal price variation of potato. Ten cold storage owners, 20 potato traders and 30 potato growers of Rangpur district in Bangladesh comprised the sample frame of the study. Both the primary and secondary data were collected. The primary data were collected through direct interview method with the help of pretested questionnaires during the months of August and September, 2011. Storing of potato in the cold storage plants certainly reduces the excessive losses of potato but all farmers can not avail the facility of cold storages due to several reasons, such as high cold storage charge, uncertainty of future market price, financial insolvency, bad communication and inadequate transport facilities and lack of any provision in getting compensation for damage of potato in the cold storage plants. Prices of potato began to increase by April and reached the peak in December. During the harvest period, potato price remained low and then it gradually rose up to the start of next harvesting period.

Validation of reference genes for expression analysis by quantitative real-time PCR in Leptinotarsa decemlineata (Say)

BackgroundL. decemlineata is an exotic invasive insect pest, and invaded in Xinjiang Uygur autonomous region in China in the 1990s from Kazakhstan. It is a notorious defoliator of potato throughout most of the northern Xinjiang in current, and often causes extremely large yield losses of potato.ResultsThe expression stability of nine L. decemlineata house-keeping genes (Actin, ACT1 and ACT2; ADP-ribosylation factor, ARF1 and ARF4; TATA box binding protein, TBP1 and TBP2; ribosomal protein RP4 and RP18; translation elongation factor 1α EF1α) was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) in seven developmental stages, three larval tissues and two insecticide treatments. The results were analyzed using three software programs: geNorm, NormFinder and BestKeeper. Although there was no consistent ranking observed among the house-keeping genes across the samples, the overall analysis revealed that RP18, RP4, ARF1, and ARF4 were the four most stable house-keeping genes. In contrast, ACT1 and ACT2, two of the most widely used reference genes, had the least stability. Our results suggest that the combined use of the four most stably expressed genes may produce optimal normalization for qRT-PCR.ConclusionsThe expression stability of the house-keeping genes varies among different developing stages, in different tissues and under different experimental conditions. Our results will enable a more accurate and reliable normalization of qRT-PCR data in L. decemlineata.

MANUFACTURE AND EVALUATION OF A LOCAL SIMPLIFIED POTATO DIGGER

Filed experiments were carried out to manufacture a simplified potato digger from locally available materials and evaluate its performance under laboratory and field conditions. Digger performance was conducted under four different soil moisture contents (d.b.) of (9, 11, 13 and 15 %) and three different digging depths of (22, 27 and 32 cm). Digging operation was carried out at four different forward speeds of (0.9, 1.6, 2.2 and 2.8 km/h). Digger performance was evaluated in terms of potato losses, digging efficiency, energy requirements and digging cost. The experimental results reveal to the following: The suitable digging depth to dig all potato tubers is 27 cm. The optimum soil moisture content suitable for digging potato is 11 %. The proper forward speed for operating the manufactured potato digger is 2.2 km/h.

First Report of Phytophthora cryptogea on Potato Tubers in Turkey.

Potato (Solanum tuberosum L.) is an important agricultural crop in Turkey, with 150,000 ha and a production of about 4 million tons per year. There are numerous pathogens that limit potato production. In 2011, a new disease was observed on the potato cultivar Ramos in Erzincan Province in Turkey. Disease symptoms were similar to pink rot. Infected tubers had a rubbery texture and when cut, infected areas were cream colored. When the infected area was exposed to air for 10 min, the color turned to salmon pink and after 15 to 20 min it turned black. A funguslike organism was isolated from diseased tubers on carrot agar. The colonies were uniform and there were no petaloid patterns. Soil extract solution was prepared using 1,000 ml of distilled water and 15 g of field soil according to Jeffers and Aldwinckle (2) and used for sporangia production. Agar discs from 5-day-old colonies were placed in soil extract solution and incubated under continuous fluorescent light at room temperature. Sporangia began forming after 24 h and were abundant after 48 h. Oogonia were not observed on corn meal agar plates supplemented by beta-cytosterol. Sporangia were nonpapillate and noncaducous, mostly ovoid, but some were obpyriform. The mean size of the sporangia was 43 ± 4 × 30 ± 2 μm. The Oomycete was identified as Phytophthora cryptogea Pethybr. & Laff., according to its morphological characters (1). Isolate identity was confirmed by sequence analysis of the ribosomal DNA internal transcribed spacer using primers ITS1 and ITS4. The ITS sequence was a 99% match to P. cryptogea strains (GenBank Accession Nos. AF087475.1, AF228101.1, GU111626.1, AY995400.1, GU111624.1, AY995400.1, and GU111624.1). The isolate from Turkey was deposited in GenBank as Accession No. JQ071495. Pathogenicity tests were conducted on potato tubers (cv. Ramos). Tubers were surface disinfected with 0.5% NaOCl for 5 min., then washed twice with sterile water. When dried, 20 tubers were wounded with a shallow cut made parallel to the surface by a sterile knife. An agar disc from a 1-week-old colony was placed on the artificial wound on each of 10 potatoes. For controls, agar discs without fungal mycelia were placed on the wounds of the other 10 potatoes. Each inoculated and uninoculated potato was incubated for 5 days at 24°C in the dark and in separate plastic cups containing moistened filter paper at the bottom. Tubers were then cut open and examined for disease. Cream colored lesions were first observed; after 10 min lesions turned salmon pink and after 20 min they turned black. Cream colored lesions were not observed inside uninoculated potatoes. To the best of our knowledge, this is the first report of P. cryptogea causing pink rot of potato in Turkey. Pink rot disease of potato is commonly caused by P. erytroseptica, but P. cryptogea can cause similar symptoms (3) and severe tuber rot. This pathogen could cause important losses of potato in Turkey, especially during storage of tubers.

Vegetative compatibility, host range and pathogenicity of Verticillium dahliae isolates in Iran

Verticillium wilt is an economically important disease which inflicts serious losses in potato, cotton, alfalfa, some vegetable crops and fruit trees and occasionally ornamentals. Verticillium dahliae, infected cultivated species and weeds were collected from several areas in Iran during tewelve years from 1993-2005 and studied for their vegetative compatibility, host range and pathogenecity. The pathogen was isolated from 27 species belonging to 24 genera and 15 families of plants but was most frequently isolated from Solanaceae, Cucurbitaceae, Oleaceae and Rosaceae hosts. The morphology of V. dahliae isolates on C and water agar media were different especially for micosclerotial appearance time (4-19 days), pigmented zone of colony (37.8-48.33 mm) and microsclerotial morphology (abundant, irregular and elongated shaped or more spherical and scattered). The ratio index of length/width of conidia ranged between 2.32 and 2.70 micrometer with an average of 2.43+0.11. Temperature influenced the radial growth ratio of the isolates and the growth response of V. dahliae isolates to temperature in vitro was quadratic. All isolates were categorized in three groups based on pathogenecity tests on differential test plants (cotton cv. Sahel and eggplant cv. local). 548 V. dahliae isolates from different locations and hosts were assigned to vegetative compatibility groups (VCGs) using nitrate-nonutilizing (nit) mutants. A higher frequency of nit1/nit3 mutants (93%) were obtained compared to nitM (7%). 51.1% of the isolates were assigned to VCG4B, 25.9% to VCG2A and 23% to VCG1. The results demonstrated that V. dahliae isolates assigned to VCGs were closely associated with specific pathogenecity within the group/diverse.

Assessment of Post-Harvest Losses of Potatoes in Bangladesh

Assessment of Post-Harvest Losses of Potatoes in Bangladesh

First Report of Sweet potato chlorotic stunt virus Infecting Sweet Potato in China.

Sweet potato chlorotic stunt virus (SPCSV) (genus Crinivirus, family Closteroviridae) is a whitefly-transmitted co-infectant of the sweet potato virus disease (SPVD), which can cause severe yield losses in sweet potato (Ipomoea batatas (L.) Lam.) (2,3). One isolate was obtained in Guangdong Province in August 2009 from sweet potato plants of cv. Guangshu No. 87 exhibiting symptoms typical of SPVD, including stunting, leaf distortion, vein clearing, and chlorosis. Leaf extracts from the symptomatic plants tested positive for SPCSV by nitrocellulose membrane ELISA with antisera specific for SPCSV obtained from the International Potato Center. Total RNA was extracted from young leaves of sweet potato and reverse transcription-PCR was performed by using primer pairs SPSP1 (5'-ATGRMTACTGRCAAAGTAAACGATG-3') and SPSP4 (5'-TCAACAGTGAAGACCRGYACCRGTCAA-3') corresponding to the capsid protein (CP) gene of SPCSV. Expected DNA fragments of 774 bp were obtained from symptomatic plants but not from control plants. The 774-bp fragments obtained by amplification were purified and cloned into the PMD19-T vector (TaKaRa, Dalian, China). Recombinant plasmids were then transformed into competent cells of Escherichia coli strain Jm109. Sequencing of the fragments from two individually clones plasmids yielded a nucleotide sequence (GenBank Accession No. HM773432) with 97.4 to 98.7% similarity to the CP gene of East African SPCSV isolates (1). To our knowledge, this is the first report of SPCSV in sweet potato fields from China. This virus may threaten sweet potato production in China, so cultivars and germplasm should be evaluated for resistance.

First report of potato blackleg caused by a biovar 3 Dickeya sp. in Georgia

In Western and Northern Europe, Dickeya species are causing increasingly severe economic losses in potato (Solanum tuberosum) crops. The costs of seed potato production resulting from Dickeya spp. infection are high due to rejection and declassification of seed tubers (Slawiak et al., 2009). Potato blackleg caused by Dickeya spp. is primarily a seed tuber-borne disease (Tsror et al., 2009). Symptoms including blackening of the stem base, wilting of plants and rotten seed tubers are observed. Recently, outbreaks of potato blackleg were recorded in Georgia on three cultivars, in Samtskhe-Javakheti region, Akhalkalaki district, in an area in excess of 100 hectares

Controlling postharvest losses of potato using biocontrol products and essential oils – an Australian perspective

Controlling postharvest losses of potato using biocontrol products and essential oils – an Australian perspective

Phytophthora erythroseptica (Pink Rot) Development in Russet Norkotah Potato Grown in Buffered Hydroponic Solutions II. pH Effects

Pink rot caused by the pathogen Phytophthora erythroseptica Pethyb. causes severe pre- and post-harvest losses in potato. Severe losses of potato from pink rot infection suggest the need for better information on cultural factors associated with disease development. Soil pH has been regarded as a strong influence on disease development as observed by previous studies and by commercial potato growers. A hydroponic growth system was established to study the effects of pH on P. erythroseptica infection and colonization in Russet Norkotah potato roots. Russet Norkotah plantlets were grown in hydroponic nutrient solutions buffered at pH 5, 6, 7, and 8 and evaluated for infection by means of quantitative Real Time PCR. Acidic pH solutions produced a higher proportion of pathogen to host DNA in root tissue, indicating increased infection and colonization at low pH. Thus, pH has a unique and significant influence on the levels of infection from P. erythroseptica and that commercially grown potatoes would likely develop reduced tuber rot if grown in soils with pH at or above 7.

Root-knot nematodes, Meloidogyne spp., on potato in Portugal

Root-knot nematodes (RKN), Meloidogyne spp., are among the most economically important nematodes in agriculture and can cause significant losses in potato (Solanum tuberosum L.), in both warm and cool climates, depending upon the nematode species (Brodie et al., 1993; Greco, 1993). RKN have not yet been recognised as serious nematode pests in the potato-growing areas of Portugal. However, during the past 10 years, nematode damage has been found in some areas and it is clear that infestation of potato fields by RKN is becoming more widespread. Some of the RKN species attacking potato, e.g., M. chitwoodi, also have quarantine status and one isolate originating from Portugal has already been included in a molecular study (Powers et al., 2005). The aim of this work was to provide data on the identification of Meloidogyne spp. on potato in Portugal. During a survey for potato cyst nematodes (PCN), Globodera spp., RKN isolates were detected in tubers and in soil collected in 210 potato fields in various districts of Portugal (Table 1). When tubers infected with RKN were detected, egg masses were collected and susceptible tomato plants (Solanum lycopersicum L.) cv. Easypeel were inoculated with ten egg masses/pot. When only soil samples were taken, tomato plants with four true leaves were transplanted into the original soil. All plants were maintained in a glasshouse at 25-30◦C. After 45 days, plants were uprooted, their roots gently washed free of soil and females, when present, collected with forceps under a stereomicroscope. Morphological characterisation was based on a minimum of 20 perineal patterns (Taylor & Netscher, 1974; Hartmann & Sasser, 1985). Their char-

Alternaria: An Identification Manual , Emory G Simmons, Centraalbureau voor Schimmelcutures, Utrecht, The Netherlands, 2007, [CBS Fungal Diversity Series No. 6.] ISBN 978-90-70351-68-7, Pp. iv + 775, figs 288. Price: 170€.

Alternaria tenuissima infects stored potatoes, and causes tuber rot, resulting in significant economic losses. As a naturally-occurring polysaccharide (poly-β-(1 → 4) N-acetyl-D-glucosamine), chitosan has been reported to be an eco-friendly alternative to synthetic fungicides for the control of postharvest diseases on agricultural commodities. In this study, application of 0.25–1.25 g/L chitosan significantly inhibited spore germination and mycelial growth of A. tenuissima in vitro, with the greatest inhibitory effect observed at the highest concentration. Cytological and biochemical analysis of A. tenuissima spores indicated that exposure to 1.25 g/L chitosan significantly damaged the plasma membrane and increased the level of lipid oxidation. Gene expression analysis in potato tuber revealed that an application of 1.25 g/L chitosan induced the expression of defense-related genes, including catalase, peroxidase, polyphenol oxidase, chitinase and β-1,3-glucanase, and the level of flavonoids and lignin. Chitosan effectively controlled tuber rot caused by A. tenuissima. Collectively, results of the current study indicate that the ability of chitosan to reduce Alternaria rot in stored potato tubers is due to its direct antifungal activity and its ability to induce defense responses in potato tuber tissues. Chitosan may have the potential as a substitute for synthetic fungicides to reduce postharvest losses in potato.

Increasing food security in Central Africa by reducing sweet potato losses due to weevils and viral diseases using biotechnology

Increasing food security in Central Africa by reducing sweet potato losses due to weevils and viral diseases using biotechnology

Spray deposition and losses in potato as a function of air-assistance and sprayer boom angle

Within the Brazilian agricultural scene, the potato crop plays a significant economic role. Modern equipment and the development of improved chemical product application techniques contribute toward the constant search for improved revenue from the potato as a crop. Field experiments were carried out in a random block design of a potato crop of the "Ágata" variety. The aim was to evaluate the effect of air assistance combined to different angles of spray application on the chemical deposition on the potato plant, as well as to assess spray losses to the soil. For this a sprayer with and without boom air assistance was used in the experiments. Boom angles of 0º, +30º and -30º were directed to a vertical position as well as forward and backward movements. The trial plants were sprayed at a volume rate of 400 L ha-1 using JA-4 hollow cone nozzles at 633 kPa and copper oxychloride as a tracer for the deposit evaluation. Trace deposits on both surfaces of leaflets were removed by washing with destilled water and were quantified by atomic absorption spectrophotometry. Spray losses were measured by placing plastic measuring collectors between rows. The deposit levels at different positions in the plant were analyzed by the Hotelling T² statistical test. Larger deposits were detected at both upper and lower positions of the plant when the spray boom was positioned at 0º and +30º in the presence of air assistance. The presence of air, in addition to the increasing spray deposits in the lower parts of the plant, helped to create greater distribution uniformity. Spray loss was below 4%.

Genetic differentiation between eastern populations and recent introductions of potato psyllid (Bactericera cockerelli) into western North America

AbstractAlthough tomato psyllid, Bactericera cockerelli (Sulc) (Homoptera, Psyllidae), annually causes significant losses in potato and tomato crops in eastern Mexico and the central United States, infestations in western North America have been historically rare. However, substantial populations appeared in 2001 in western North America and caused losses in tomato production exceeding 80%; losses in 2004 reached 50%. To determine if these new outbreaks were the result of a simple range expansion or the evolution of a new B. cockerelli biotype, inter simple sequence repeat (ISSR) markers, as well as mitochondrial gene cytochrome oxidase I (COI), internal transcribed spacer 2 (ITS2), and wsp sequence data were used to characterize populations of the psyllid. Western populations from Baja, Mexico, Orange County, and Ventura County were compared with populations from central USA (Colorado and Nebraska) and eastern Mexico (Coahuila). Based on ISSR markers, the psyllid populations clustered into two groups, with one group including populations from western North America and the other group including populations from central USA and eastern Mexico. For COI comparisons, there was one base‐pair difference found in the 544 bp‐long COI fragments, but the populations again segregated along the same geographic lines. Two strains of Wolbachia were identified, the maximal differences between wsp clones from all populations was 5 bp for strain Bac1 and 23 bp for strain Bac2 out of a 555‐bp fragment. The ISSR data, therefore, were consistent in indicating the development of a new psyllid biotype that has adapted to western North America rather than a simple range expansion, but the other genetic data sets were less conclusive.

Late Blight of Potato and Tomato in the Genomics Era.

Tremendous progress in the availability of genomic data has been made in the past decade. With the advent of high throughput and affordable DNA sequencing, the entire hereditary blueprint of an organism can be determined. A widespread transition into research enabled by the tools and resources of genomics is consequently occurring, and it has already affected countless disciplines within the biological sciences, including plant pathology. This new era of research allows a comprehensive study of gene structure and function and has already delivered tremendous insights into the basic biology of living organisms. Genomics offers new opportunities for applied biological sciences and is poised to impact plant health and plant disease management. More than 150 years have elapsed since Phytophthora infestans caused the Irish potato famine, but strategies for managing potato and tomato late blight often remain unsustainable and costly. In effect, P. infestans continues to cost billions of dollars annually through losses in potato and tomato production and increased fungicide costs (15,45,47,48). In the United States and other developed countries, chronic use of chemicals to manage late blight reduces the profit margins of farmers and is not always successful. Most populations of the pathogen are resistant to metalaxyl (and the active enantiomer mefenoxam), which had previously been very successful for controlling P. infestans (19,47). In developing countries, late blight also affects subsistence potato production. Recent epidemics in the 1990s, notably in eastern Europe, have caused significant losses in yield (20,46). In 2003, potato production was nearly eliminated in Papua New Guinea, one of the few countries in the world that was previously free of the disease (4). Remarkably, the disease spread through the entire country within 2 months of first incidence. Disturbing reports predict that potato late blight will continue to cause food shortages and hunger in several parts of the world (20,46).

The impact of tropospheric O3 on leaf number duration and tuber yield of the potato (Solanum tuberosum L.) cultivars Bintje and Kardal

Ozone (O3) is a major phytotoxic air pollutant with the potential to cause severe yield losses in potato (Solanum tubersosum L.) and in other crops. The present study was aimed (i) to investigate the O3 sensitivity of two potato cultivars (Bintje and Kardal) in relation to their earliness in maturation, and (ii) to analyse possible O3 effects on potato tuber dry mass (DM) production in relation to leaf duration. The experiment was performed in the southwest of Sweden using open-top chamber (OTC) technique. The crops were exposed to three levels of O3: charcoal-filtered air (CF), non-filtered air (NF) and non-filtered air + 26 nmol mol−1 O3 (NF+). In addition, ambient air plots were used to monitor the impact of the OTC enclosure. Leaf number duration (LND) was calculated as the number of leaves integrated over the temperature sum accumulation during the season. In the present study, the O3 effects on potato were characterised by visible injury of the leaves and earlier leaf senescence. The haulm/plant DM ratio was significantly lower in NF+ compared to CF and NF. The conclusions were (i) that the early to intermediate maturing cultivar Bintje tended to suffer more from O3 injury than the late maturing cultivar Kardal, (ii) that this was due to a difference in the ability to compensate for haulm damage by the development of new leaves rather than to a difference in leaf O3 sensitivity, and (iii) that a rather low O3 exposure can induce a significant reduction in LND in potato. The O3 effects on tuber DM (−2% in NF+ and +31% increase in CF, both compared to NF) could not be statistically demonstrated, although there was a strong correlation between LND and tuber DM.