Impact of carrot resistance on development of the <Emphasis Type="Italic">Alternaria</Emphasis> leaf blight pathogen (<Emphasis Type="Italic">Alternaria dauci</Emphasis>)

The interaction between Alternaria dauci and two carrot cultivars differing in their resistance to leaf blight was investigated by microscopy. The fungal development between 1 and 15 days post-inoculation was quite similar in the susceptible cv. Presto and the partially resistant cv. Texto: After conidial germination, leaf adhesion of the pathogen was achieved with mucilaginous filaments; hyphae penetrated the leaves directly with/without the formation of appressoria-like structures or via stomata; the fungus spread by epiphytic hyphae with hyphopodia and subcuticular mycelia. Intense necrotic plant cell reactions occurred under the fungal structures. At 21 days post-inoculation, typical features of fungal development were noted for each cultivar: growing hyphae emerged from stomata in cv. Presto, whereas conidiophores without conidia were observed in cv. Texto. Leaf tissues of both cultivars were strongly damaged and vesicle-like structures (assumed to be plant phenolics) were abundantly present between mesophyll cells. A real-time PCR method was developed for in planta quantification of A. dauci. Between 1 and 15 days post-inoculation, the fungal biomass was equivalent in the two cultivars and was about fourfold higher in cv. Presto than cv. Texto at 21 and 25 days post-inoculation. Taken together, our results indicated that A. dauci was able to colonize both cultivars in a similar manner during the first steps of the interaction, then fungal development in the partially resistant cultivar was restricted due to putative plant defence reactions. The results of this study enhance the overall understanding of infection processes in the A. dauci-carrot pathosystem.     

Fusarium solani f. sp. passiflorae: a new forma specialis causing collar rot in yellow passion fruit

The aim of this study was to characterize a Fusarium population obtained from yellow passion fruit (YPF) with collar rot using pathogenicity, morphocultural characteristics and molecular tests. Pathogenicity and disease severity were assessed in six plant species: YPF, zucchini, tomato, bean, soya bean and cucumber. Potato dextrose agar medium (PDA) was used to determine mycelial growth at five temperatures (15–35°C). The colour produced by isolates was also determined on PDA at 25°C. Synthetic nutrient agar medium was used to evaluate: (i) type of mycelium and phialides; (ii) size, shape and number of septa from conidia; and (iii) production of chlamydospores and perithecia. Molecular tests consisted of sequencing the ITS–5·8S rDNA region and elongation factor 1α (EF‐1α) gene. The isolates caused large lesions on YPF, zucchini and tomato, with YPF having the highest mean disease severity and being the only one that showed wilt symptoms and death of the plant. Thus the isolates showed host specificity. Maximum mycelial growth occurred at 25°C and the predominant colour was bluish‐white. The isolates produced long phialides, dense aerial mycelium, oval microconidia with a mean size of 9·5 × 2·6 μm, macroconidia of 32·7 × 3·4 μm with 3·3 septa, and chlamydospores; only one isolate lacked perithecia. Phylogenetic trees of the ITS region and EF‐1α gene showed that isolates from YPF formed a distinct group within the F. solani group and the formae speciales of F. solani. It is proposed to name all isolates from YPF as F. solani f. sp. passiflorae.     

An optimized method for in vitro production of Verticillium dahliae microsclerotia

Verticillium dahliae causes wilt disease of many crops worldwide. Microsclerotia are the main resting structure of V. dahliae in soil and can survive for more than 10 years, serving as an important source of primary inoculum. Mass production of microsclerotia in laboratory is valuable for studying various aspects of V. dahilae, such as its biology, epidemiology and control. We failed to produce a sufficient amount of microsclerotia in vitro for one strain of our interest using several previously published methods. Therefore, we developed a new protocol for mass in vitro production of viable microsclerotia. Verticillium strains were cultured in a modified basal agar medium at pH of 11.5 under 20 °C and incubated for 25 days in dark. When 16 strains were subjected to this condition, large numbers of microsclerotia were produced although varied greatly among strains, including the strain that failed to produce microsclerotia with previously published methods. Microsclerotia from 14 of the 16 strains resulted in wilt development on inoculated cotton seedlings.     

Sensitivity to chloropicrin of microsclerotia ofVerticillium dahliae as related to their germination on growth media and in soil

Sensitivity of microsclerotia ofVerticillium dahliae to chloropicrin increased considerably during incubation on Czapek’s medium and in moist soil. However, whereas on a growth medium maximum sensitivity was obtained after 48–96 h, a period of 216–288 h was required to obtain a similar degree of sensitivity of microsclerotia incubated in moist soil. At the end of the incubation period, the microsclerotia which had been incubated in moist soil gave rise to an average of four hyphae per microsclerotium as compared with 130 observed on Czapek’s agar. It is concluded that germination does not play a major role in the increase of sensitivity to chloropicrin during the incubation of microsclerotia ofV. dahliae in moist soil.     

拮抗性链霉菌对大丽轮枝菌微菌核形成与萌发的影响

为探索拮抗性链霉菌对棉花黄萎病病原大丽轮枝菌Verticillium dahliae Kleb.的抑菌机制,采用菌丝生长速率法、微菌核萌发法研究了6株拮抗链霉菌无菌发酵滤液对大丽轮枝菌生长、微菌核形成与萌发的影响。链霉菌无菌发酵滤液对大丽轮枝菌菌落生长、菌核形成和微菌核萌发有明显抑制作用。其中菌株B49的抑菌效果最好,5倍稀释发酵液培养14天时对菌落生长的抑菌率达69.7%;菌株B49、D184和Act12的5倍稀释发酵液对微菌核形成的抑制率达100%;将经B49、D184和Act12发酵液处理后丧失形成微菌核能力的大丽轮枝菌菌株转接至不含发酵液的PDA培养基,连续传代至第5代,其仍然不能恢复形成微菌核的能力;微菌核在含有菌株D184 5倍稀释发酵液的培养基上培养168 h时,萌发率仅为38.3%。     

The overwintering ofExserohilum turcicum in Israel

Exserohilum turcicum (Pass.) Leonard and Suggs, the causal agent of northern leaf blight of corn, overwinters onSorghum halepense L. plants and on corn debris (dead leaves). Spqrulating lesions ofE. turcicum were observed on sorghum plants in the winter (February). Spores from these lesions were pathogenic to susceptible sweet corn plants cv. ‘Jubilee’. Infected sporulating leaves of corn were stored for 1 year at 20°C (40-60% relative humidity), at 5°C (60% relative humidity), or buried 5 cm underground. During the storage period, 32% and 22% of the spores formed chlamydospores, at 20° and 5°C, respectively. Leaves buried 5 cm underground were totally decomposed after 6 months. After 4 months, 25% of the spores in the buried leaves had formed chlamydospores. Spores with chlamydospores were pathogenic to corn plants. The viability of spores without chlamydospores stored at 20°, 5°C or buried underground was 0, 60 and 0%, respectively. In a parallel experiment infected leaves were stored under similar conditions and allowed to sporulate. No sporulation occurred on infected leaves buried in soil. Spores produced on infected leaves stored at 20° and 5°C were highly pathogenic to corn plants. In leaves treated with 0.1N glucose, chlamy dospore formation was significantly inhibited.     

Microscopy of invasion of red clover roots by Trichocladium basicola, and effects of benomyl and prochloraz

Roots of red clover seedlings grown on plates of water agar, or water agar containing benomyl or prochloraz, were inoculated with conidia of Trichocladium basicola and examined by light and transmission electron microscopy. Penetration of host epidermal cells occurred from about 16 h after inoculation of untreated or fungicide-treated seedlings. Intracellular hyphae were constricted at septa and had a beaded appearance. They invaginated the host plasmalemma, but had no obvious deleterious effect on the cytoplasm until they had grown to fill much of the lumen, when host cells degenerated and died. As colonization of the cortex progressed, straight, unconstricted hyphae were formed and from these reproductive hyphae developed, which produced endoconidiophores and chlamydospores on the root surface. Penetration of host cell walls appeared to involve localized action of fungal enzymes. Papillae were often found at sites of penetration, but these rarely obstructed fungal development. Seedlings treated with prochloraz had fewer sites of fungal penetration, and fewer cells in the beaded hyphae than untreated seedlings or those treated with benomyl. Both fungicides caused abnormalities in fungal ultrastructure. Hyphae treated with benomyl were often found to contain lomasomes, while those treated with prochloraz had thickened, fragmented walls, and disorganized cell contents.     

Temperature adaptation in isolates of Sclerotinia sclerotiorum affects their ability to infect Brassica carinata

Sclerotinia stem rot (Sclerotinia sclerotiorum) is a serious disease in oilseed Brassica crops worldwide. In this study, temperature adaptation in isolates of S. sclerotiorum collected from differing climatic zones is reported for the first time on any crop. Sclerotinia sclerotiorum isolates from oilseed rape (Brassica napus) crops in warmer northern agricultural regions of Western Australia (WW3, UWA 7S3) differed in their reaction to temperature from those from cooler southern regions (MBRS‐1, UWA 10S2) in virulence on Brassica carinata, growth on agar, and oxalic acid production. Increasing temperature from 22/18°C (day/night) to 28/24°C increased lesion diameter on cotyledons of B. carinataBC054113 more than tenfold for warmer region isolates, but did not affect lesion size for cooler region isolates. Mean lesion length averaged across two B. carinata genotypes (resistant and susceptible) fell from 4·6 to 2·4 mm for MBRS‐1 when temperature increased from 25/21°C to 28/24°C but rose for WW3 (2·35 and 3·21 mm, respectively). WW3, usually designated as low in virulence, caused as much disease on stems at 28/24°C as MBRS‐1, historically designated as highly virulent. Isolates collected from cooler areas grew better at low temperatures on agar. While all grew on potato dextrose agar between 5 and 30°C, with maximum growth at 20–25°C, growth was severely restricted above 32°C, and only UWA 7S3 grew at 35°C. Oxalate production increased as temperature increased from 10 to 25°C for isolates MBRS‐1, WW3 and UWA 7S3, but declined from a maximum level of 101 mg g^?1 mycelium at 20°C to 24 mg g^?1 mycelium at 25°C for UWA 10S2.     

Evaluation of different methods for the characterization of carrot resistance to the alternaria leaf blight pathogen (Alternaria dauci) revealed two qualitatively different resistances

Alternaria leaf blight (ALB), caused by Alternaria dauci, is one of the most damaging foliar diseases of carrot worldwide. The aim of this study was to compare different methods for evaluating levels of carrot resistance to ALB. Three techniques were investigated by comparison with a visual disease assessment control: in vivo conidial germination, a bioassay based on a drop‐inoculation method, and in planta quantification of fungal biomass by quantitative PCR (Q‐PCR). Three carrot cultivars showing different degrees of resistance to A. dauci were used, i.e. a susceptible cultivar (Presto) and two partially resistant genotypes (Texto and Bolero), challenged with an aggressive or a very aggressive isolate of A. dauci. Both partially resistant genotypes produced a higher mean number of germ tubes per conidium (up to 3·42±0·35) than the susceptible one (1·26±0·18). The drop‐inoculation results allowed one of the partially resistant genotypes (Bolero, log₁₀(S+1) = 1·34±0·13) to be distinguished from the susceptible one (1·90±0·13). By contrast, fungal growth measured by Q‐PCR clearly differentiated the two partially resistant genotypes with log₁₀(I) values of 2·77±0·13 compared to the susceptible cultivar (3·65±0·13) at 15 days post‐inoculation. This result was strongly correlated (r² = 0·91) with the disease severity index scored at the same date. Data obtained with the different assessment methods strongly suggest that the Texto and Bolero genotypes have different genetic resistance sources.     

Cultural characteristics of Sporisorium sorghi and detection of the pathogen in plant tissue by microscopy and polymerase chain reaction

Despite the economic importance of covered kernel smut of sorghum (Sporisorium sorghi) in many African states and other parts of the world, only limited information is available on laboratory cultivation methods for this fungus and techniques for its diagnosis in plant tissue. The current paper describes laboratory and greenhouse experiments performed with field material of S. sorghi. When intact sori were kept at 5°C, 80% of the spores germinated even after 24?months of storage. Spore germination on agar medium and production of mycelial dry weight in still culture were highest between 20° and 35°C, with a peak at 30°C. Both showed a steady increase from pH 4.5 to pH 7.5, followed by a decline at pH 8.5 and 9.5. In shake culture in different broth media the addition of 0.3% peptone from soybean caused an increase in fungal growth compared with the media alone. Of the media tested, mycelial production was highest in malt dextrose broth supplemented with peptone. When cultivated on different agar media, the morphology of single spore isolates differed both among isolates and depending on the agar medium. In greenhouse experiments, five short, early maturing sorghum breeding accessions proved to be partially or fully resistant to covered kernel smut. Among the plant materials tested, cv. ??Dorado?? appeared to be the one best suited for greenhouse experiments with covered kernel smut. By microscopy of hand-cut sections stained with trypan-blue, hyphae of S. sorghi were seen in apical buds and in nodes of young sorghum plants. Diagnostic PCR amplified a 903?bp element comprising the internal region of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoding gene and enabled the detection of S. sorghi in both nodes and apical buds of infected sorghum seedlings. Both techniques, i.e., microscopy and diagnostic PCR, have the potential to be used in studies for the identification of effective sorghum seed treatments already at the seedling stage.     

Black Streak of Edible Burdock Caused by Itersonilia perplexans in Japan

Black streak disease of edible burdock (Arctium lappa L.) has been observed periodically in Hokkaido Prefecture, Japan since 1988. Symptoms appeared initially as small, dark brown to black spots on the leaf veins and petioles. The necrotic spots developed longitudinally along the leaf veins or petioles. Diseased leaf veins or petioles occasionally snapped off at the necrotic lesions. An Itersonilia sp. was isolated from rotting leaf veins and petioles. Laboratory inoculations of edible burdock seedlings using ballistospore suspensions produced typical symptoms observed in nature. The fungus had a feathery mycelium and developed a white to pale cream colony color. The mycelium was composed primarily of branched hyphae with clamp connections at the septa. Ballistospores, formed at the apex of inflated cells, were lunate, ovoid to pyriform. The fungus occasionally produced appressoria, chlamydospores and yeast cells. Based on the morphological characteristics, the causal agent was identified as Itersonilia perplexans Derx. Edible burdock strains were also pathogenic to chrysanthemum and caused petal blight. This report is the first of a foliar disease of edible burdock caused by I. perplexans in Japan. Received 8 April 2002/ Accepted in revised form 26 June 2002     

<Emphasis Type="Italic">Fibulorhizoctonia psychrophila</Emphasis> is the causal agent of lenticel spot on apple and pear fruit in the Netherlands

In a survey for postharvest diseases of apples and pears in the Netherlands, an unknown postharvest fruit rot was observed. The disease appeared to originate from infected lenticels. A fungus was consistently isolated from the decayed fruits. The fungal pathogen was isolated on potato dextrose agar, and at low temperatures development of a fast-growing whitish mycelium was observed. Growth of this fungus was observed between 1 and 20 °C with an optimum at 15 °C, while incubation of mycelium at 25 °C resulted in no growth. The isolates did not produce asexual or sexual spores. The isolates were characterized and identified by morphology and molecular phylogenetic analysis. Genomic DNA was isolated and amplified using ITS1-ITS4, EF1 and RPB2 primers, and BLAST searches in GenBank placed the fungus taxonomically in the genus Fibulorhizoctonia, with the highest matches to F. psychrophila. Pathogenicity of representative isolates from apple and pear fruit was confirmed under laboratory conditions. To the best of our knowledge this is the first report of F. psychrophila causing lenticel spot on apple and pear, and also the cause of a whitish mould on storage bins.     

Effect of plant roots on the germination of microsclerotia ofVerticillum dahliae

Root observation boxes were used to study the effects of hosts and non-hosts on the germination of microsclerotia ofV. dahliae. The effects of roots on microsclerotia were examined within a radius of 1 mm around the root tip. Host plants such as potato and field bean induced a higher percentage of germination of the microsclerotia than a non-host such as barley. A susceptible potato cultivar stimulated germination more than a resistant cultivar. The germination percentage and the number of hyphae per microsclerotium decreased with distance from the root surface regardless of the plant species or cultivar.     

A selective medium for the detection ofAlternaria dauci andAlternaria radicina

A selective medium to detectAlternaria dauci andA. radicina on seed, plant debris and other substrates was developed. Growth and sporulation by most undesired organisms was reduced, but adequate mycelial growth and sporulation byA. dauci andA. radicina was maintained so they could be identified by their unusual mycelial growth or characteristic spores. The medium is based upon carrot leaf extract, which promotes profuse sporulation by both pathogens. Glucose, sodium polypectate and mineral salts further enhanced spore and mycelial production. Streptomycin sulfate and metalaxyl (or mefenoxam), combined with either benomyl or thiophanate-methyl, reduced growth and sporulation of unwanted organisms. There were strong interactions among the fungicides, bactericide, and most other medium components. There were also significant effects on the type of mycelial growth produced by some medium components. If the unique, dark mycelia produced by both pathogens is the desired detection method, the medium components must be optimized for eitherA. dauci orA. radicina because the two fungi responded differently in this regard. All medium configurations allowed both fungi to be identified by their characteristic spores. The sensitivity of theAlternaria dauci-radicina selective medium (ADRSM) to detectA. radicina on carrot seeds was similar to other methods presently in use, but ADRSM was more sensitive than the other methods for detectingA. dauci on infested carrot seeds.     

Influence of rate of soil fertilization on alternaria leaf blight (Alternaria dauci) in carrots

The possibility of suppressingAlternaria dauci (Kühn) Groves & Skolko, the causal agent of Alternaria leaf blight in carrot, by excess application of fertilizer was examined in greenhouse and field experiments. Reducing the rate of fertilization by one half from the optimal rate (100 ppm N, 19 ppm P and 74 ppm K) resulted in a 23–30% increase in the severity of Alternaria leaf blight. However, doubling the rate of fertilization resulted in only a 10–15% decrease in disease severity. Inoculating with different concentrations ofA. dauci spores (10³ or 10⁴ spores/ml) did not alter the response of the plants to the fertilization rate, although significantly higher disease severity was observed in plants inoculated with the higher spore concentration. These results were corroborated in the field, where neither disease severity nor harvested yield was significantly affected by tripling the amount of soil fertilization. Application of foliar fungicides, on the other hand, had substantial effects on both disease and yield. Therefore, it was concluded that carrot crops should be fertilized and maintained for optimum yield, and thatA. dauci should be managed by properly timed applications of fungicides during the growing season. Contribution no. 533/99 from the Inst. of Plant Protection, Agricultural Research Organization.     

Inactivation of Phytophthora and bacterial species in water by a potential energy-saving heat treatment

Plant pathogens, especially Phytophthora and bacterial species, in re-circulated irrigation water present a significant health risk to nursery and greenhouse crops. Heat treatment at 95°C for 30?s is one of the most reliable technologies for irrigation water decontamination. The primary objective here was to examine whether the water temperature required to inactivate major pathogens in re-circulated irrigation water can be lowered from 95°C to conserve energy and improve horticultural profitability while reducing environmental footprint. Specifically, we investigated the effect of water temperature on Phytophthora nicotianae zoospore survival in the laboratory and on annual vinca under greenhouse conditions. We also assessed the effect of water temperature on survival of chlamydospores of P. nicotianae, oospores of P. pini, six plant pathogenic bacterial species and Escherichia coli. The zoospores of P. nicotianae did not survive and cause any disease on annual vinca when exposed to 42°C for 12?h or 48°C for 6?h. No chlamydospores of P. nicotianae survived 42°C for 24?h or 48°C for 6?h, nor did the oospores of P. pini at 42°C for 12?h or 48°C for 6?h. In addition, none of the seven bacterial species survived 48°C for 24?h. These results indicate that the required water temperature to eliminate Phytophthora and bacterial species may be lowered substantially from 95°C by longer exposure time, improving the economics and environmental footprint, without sacrificing efficacy of heat treatment.     

Long‐term viability of the northern anthracnose pathogen,Kabatiella caulivora,facilitates its transportation and spread

The conidia and resting hyphae of the northern anthracnose pathogen of Trifolium species, Kabatiella caulivora, were effectively carried by, and maintained long‐term viability on, a range of materials, including metals, fabrics, woods and plastics. Conidia and hyphae became thick‐walled and melanized with time. There were significant (P < 0.001) differences in conidia/resting hyphae survival between carrier materials and between temperature regimes. At 23 °C/8 °C day/night, conidia and resting hyphae remained viable on steel, corrugated iron, galvanized steel, all tested fabrics, wood and random mixed materials for up to 8 months. At 36 °C/14 °C day/night, conidia and resting hyphae remained viable for up to 8 months, but only on cotton, denim, fleece, silk, leather, paper, plastic and all wood materials. At 45 °C/15 °C day/night, conidia and resting hyphae remained viable up to 8 months only on fleece wool, Eucalyptus marginata (jarrah wood) and paper. There were significant differences between carrier materials in their abilities to retain conidia and resting hyphae after washing (P < 0.001). Metabolic activity was confirmed for conidia and resting hyphae recovered after 8 months and K. caulivora colonies successfully re‐established on potato dextrose agar. Findings confirmed the critical importance of materials as long‐term carriers of viable K. caulivora conidia and resting hyphae, highlighting the potential for spread of a highly virulent K. caulivora race within and outside Australia via farming equipment, clothing and other associated materials. Results also have wider biosecurity implications for the transportation of fungal‐infested carrier materials previously considered as low risk.     

百合疫病病原菌的鉴定及培养基的筛选

从具典型症状的新鲜百合疫病植株茎基部病组织中分离到百合疫霉菌,根据其病原菌菌丝的形态、菌落特征,厚垣孢子、游动孢子囊和卵孢子的形态和大小,以及病原菌致病性测定,该病原菌鉴定为烟草疫霉Phytophthora nicotianae van Brede de Haan.供试的16种培养基中,病原菌在胡萝卜琼脂培养基(CaA)和辣椒琼脂培养基(PeA)上生长最好,生长速率分别为1.771和1.770mm/h.在常规培养条件下,病原菌不易产生厚垣孢子、游动孢子囊和卵孢子,在低温、皮氏溶液和土壤浸出液中分别诱导产生出大量厚垣孢子、游动孢子囊和卵孢子.     

Alternaria eichhorniae, a biological control agent for waterhyacinth: mycoherbicidal formulation and physiological and ultrastructural host responses

The bioherbicidal efficacy of different alginate formulations of Alternaria eichhorniae 5 (isolate Ae5), a virulent Egyptian isolate, was compared on waterhyacinth (Eichhornia crassipes). The fungus was formulated as alginate pellets containing mycelium alone, mycelium plus culture filtrate or culture filtrate alone. Each formulation was applied with and without a hydrophilic humectant (Evergreen 500). These formulations were evaluated for disease incidence (DI), and disease severity (DS). Maximum DS, but not DI, was obtained with the alginate pellets of mycelium plus culture filtrate. Alginate formulations supplemented with the hydrophilic polymer were more effective in promoting disease. Physiological changes associated with the treated waterhyacinth plants were determined 3, 6 and 9 days after treatment. Waterhyacinth plants treated with alginate pellets of mycelium plus culture filtrate of Ae5 had the lowest levels of pigments, carbohydrates and relative water content. Infection of waterhyacinth with Ae5 led to a significant increase in total phenols of leaves as compared to control. Penetration of waterhyacinth leaves by the fungus occurred only through the stomata, and the invading hyphae were located in the intercellular spaces of leaf tissues. Cytological changes noted in infected cells included changes in chloroplast, nucleus and mitochondria. Invagination of the plasma membrane, particularly at plasmodesmata was also noticed in infected cells. The associations between the infection process, the physiological disorder and the ultrastructure of infected leaves are discussed.     

Cytological and biochemical changes induced by chitosan in the pathosystem Alternaria alternata-tomato

The cytological and biochemical response of the fungus Alternaria alternata to chitosan application in tomato fruits was evaluated. The research was developed in the following stages: microscopically to observe the degree of damage that chitosan causes over the conidia and hyphae of the fungus at the structural level and during the infection process in tomato tissue. Biochemically we tried to identify the elicitation of the phytoalexin rhisitin and other compounds involved in resistance. At the microscopic level, mycelium and conidia of chitosan-treated of A. alternata showed cell wall disintegration, plasma membrane retraction, cellular distortion, release of the apical portion of the conidia and lysis of fungal cells. Hyphae and conidia were susceptible to chitosan application. Infection always took place in chitosan treated and inoculated tomatoes and it was difficult to observe ultrastructural alterations due to chitosan application. The phytoalexin rhisitin was not isolated from any of the treatments but other compounds such as alkenes, fatty acids and vitamin E whose antimicrobial effects have been reported were detected. The elicitation of precursor compounds in the pathosystem A. alternata-tomato was more associated with the infection process than with the chitosan application. Further studies are necessary to confirm these findings.