Carrot Root Slices Research Articles

First Report of Ceratocystis changhui Causing Postharvest Fruit Rot of Peach in Kunming, China.

The peach (Prunus persica (L) Batsch) is a predominant commercially grown stone fruit in China (Lee et al. 1990). Ceratocystis changhui is an aggressive pathogen causing typical black rot symptoms on corms of taro (Colocasia esculenta) (Liu et al. 2018), it has not been reported on other hosts. During the summer and autumn of 2013, a postharvest fruit rot disease was observed on several peaches at a farmer's market (N 25°02'; E 102°42') in Kunming City, Yunnan Province, China. The incidence of the disease varied from 5 to 20%. Necrotic spots were first observed on the infected peach fruit (Prunus persica cv. shuimitao). The spots enlarged gradually and developed into a brown, water-soaked and rotted lesion. Eventually, the whole fruit became soft, rotted and covered with a gray-brown mycelium (Fig. 1 A, B). The isolates were obtained from the symptomatic tissues incubated on slices of fresh carrot root (Moller et al. 1968). After 5 to 10 days of incubation, perithecia and mycelium were observed growing on carrot slices. Spore masses were removed from the apices of perithecia, transferred to potato dextrose agar medium (PDA) and incubated at 25°C for 5 to 10 days, followed by single-spore isolation. All eight single-spore isolates from peach fruits obtained in this study were deposited in the State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, China. In culture, mycelium was initially white, gradually turned to greyish-green or brown (Fig. 1E, F). Measurements were made 7 days after the formation of perithecia. Perithecia (Fig. 1G) were black, globose, 185.71 to 305.56 μm × 142.86 to 264.29 µm and showed a long black neck, 600 to 957.14 µm (Fig. 1H). Ascospores (Fig. 1I) were helmet-hat shaped and 2.86 to 6.67 µm ×3.81 to 4.76 µm. Cylindrical conidia (Fig. 1J) 6.67 to 38. 95 µm × 2.86 to7.62 µm were observed. Chlamydospore (Fig. 1K), 8.57 to 13.33 μm × 5.71 to 9.52 μm, were ovoid or obpyriform, smooth. To further verify pathogen identity the internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS1F and ITS4 (Thorpe et al. 2005), and the total genomic DNA from the mycelia of five isolates was extracted using a CTAB method (Lee &Taylor 1990). The nucleotide sequences have been blasted and deposited in the GenBank database. Analysis of the ITS sequences from the isolates T1-1yp, T1-2yp, T2-1yp (GenBank accession no. KY580895-KY580897) showed 99% to 100% similarity with isolates C. changhui CMW43272 (KY643886), CMW43281 (KY643884), CMW46112 (KY643891) and CMW46113 (KY643892) from taro in China. Phylogenetic trees based on the maximum-likelihood (ML) method were constructed using MEGA 7. ITS sequences of other Ceratocystis spp. were attained from NCBI for comparative analysis (Liu et al. 2018), and Davidsoniella virescens (CMW11164) served as outgroup. The robustness of ML tree was evaluated with 1,000 bootstrap (BS) values. The pathogen was identified as C. changhui based on the phylogenetic analysis (Fig. 2). Three isolates (T1-1yp, T1-2yp, T2-1yp) were used for pathogenicity. Nine Prunus persica cv. yingzuitao fruits at early maturity (8 points out of 10) were wound inoculated with 200μL conidia suspension of the fungus (approximately 2.0 × 106 conidia / mL). Degreasing cotton dipped in sterile water was used to raise the humidity in preservation boxes. Boxes were incubated for 10 days at 25°C. Three peaches as controls were treated only with sterile distilled water in the same way. Symptoms of sunken lesions and fruit rot were observed two days after inoculation, and measured at 1.8 to 3.2 cm from the inoculation point within 5 days (Fig. 1C: right, D). The same pathogen was re-isolated from them confirming Koch's postulates. Control peaches remained symptomless. This fungus was morphologically and phylogenetically identified as C. changhui. To our knowledge, this is the first report of C. changhui on postharvest peach in Yunnan, China. The disease will affect quality and taste of peach, so it is critical to deploy appropriate management strategies to limit the fungus spread.

First Report of Ceratocystis fimbriata Causing Postharvest Fruit Rot of Cucumber in China

First Report of <i>Ceratocystis fimbriata</i> Causing Postharvest Fruit Rot of Cucumber in China

Transcriptional regulation of chlorogenic acid biosynthesis in carrot root slices exposed to UV-B light

Orange carrots are well known for their nutritional value as producers of β-carotene, a vitamin A precursor. Lesser known, is their ability to accumulate antioxidants such as chlorogenic acid. Chlorogenic acid is produced through the same biosynthetic pathway that produces lignins, anthocyanins, flavonols and isoflavonoids, the phenylpropanoid pathway. This pathway can be induced by UV-B light and wounding to produce large amounts of chlorogenic acid. We identified a number of the carrot structural genes in this pathway and their proposed regulators in carrot root slices and analyzed their expression in response to UV-B light exposure using real-time PCR. Our results indicate that not only does the expression of the structural genes correlate with their proposed regulators, including DcHY5 and others, but also correlate with chlorogenic acid production. Our results indicate that the HY5 — UVR8 signaling network may exist and function in carrot roots.

First Report of Wilt of Eucalyptus Caused by Ceratocystis fimbriata in China.

Eucalyptus spp., most of which are native to Australia, are one of the world's most commonly planted forestry crops, and have been widely planted in the tropics and subtropics. Eucalyptus trees are distributed in more than 600 counties in 19 provinces/regions throughout China, especially in the southern regions of the country (1). From April of 2013 to May of 2014, eucalyptus trees were noted to be wilting and dying rapidly in Kunming, Yunnan Province (25°02' N, 102°42' E), southwest China. Symptomatic trees typically have many wilted or dead leaves hanging throughout the tree or on some major branches, sometimes followed by death of the whole tree. Dark brown to black discoloration can be seen in the woody xylem, and elongated cankers were also present on some affected trees. A fungus was consistently isolated from the leaves and cankers when symptomatic tissue was incubated between two slices of fresh carrot root. Spore masses were removed from the apices of perithecia, transferred to malt extract agar medium (MEA), and incubated at 25°C. Perithecia developed on the medium, and were black, globose, 212.5 to 242.4 × 207.5 to 254.2 μm, and possessed a long black neck (952.3 to 1,303.3 μm). Ascospores had the typical "hat" morphology and were 4.3 to 5.2 × 3.0 to 3.8 μm. Cylindrical endoconidia (11.2 to 22.2 × 3.9 to 6.1 μm) were found. Chlamydospores produced on media were ovoid and smooth, and were 7.8 to 9.7 × 9.9 to 12.8 μm. Chains of barrel-shaped conidia were not found. PCR amplification and sequencing of the ITS region of rDNA were carried out for one isolate, E2-2, using the procedures of Thorpe et al. (3). Analysis of ITS sequence data (GenBank Accession No. KJ511481) showed that the isolates were 99% homologous to the isolate of C. fimbriata from diseased Colocasia esculenta in Cuba, China, and Hawaii (AY526304 to 06) by BLAST analysis. Thus, the fungus was identified as C. fimbriata based on morphological and molecular characteristics. Pathogenicity tests were made on 1-year-old E. grandis seedlings as follows. A conidial suspension of each isolate was diluted to 106 conidia/ml, and 0.2 ml was injected into wounds on three petioles on each of five healthy plants of E. grandis, and control seedlings were injected with sterile water that had been placed on MEA plates. The seedlings were incubated at 25°C in moist chambers. After 3 days, all inoculated E. grandis plants showed dark brown to black discoloration in the leaf axils. After 5 days, leaf wilting was present. C. fimbriata was re-isolated from all inoculated symptomatic tissue. No symptoms were visible in the control plants and no fungus was isolated from them. Ceratocystis wilt was first observed in eucalyptus in 1997 in the state of Bahia. This was followed by a report of C. fimbriata causing wilt of E. grandis in the Republic of Congo, Uganda, and Uruguay (2). Chen et al. reported two species of Ceratocystis, C. acaciivora and a previously undescribed species C. chinaeucensis, from eucalyptus plantations in Guangdong Province in China (1). To our knowledge, this is the first report of C. fimbriata causing wilt of eucalyptus in China.

First Report of Ceratocystis fimbriata-Caused Wilt of Eriobotrya japonica in China.

The loquat (Eriobotrya japonica (Thunb.) Lindl.), an important perennial evergreen fruit crop that originated in China and is also cultivated as an ornamental plant, is well known all over the world for high nutritional, medicinal, economic, and ecological values (4). So far, it has been grown in more than 30 countries (1) and is becoming an important industry in China, Spain, Japan, India, Pakistan, and Turkey (2). During an investigation from May to August of 2013, severely withered loquat plants were observed in Kunming city of Yunnan Province (25°02' N; 102°42' E), Southwest China. Initial symptoms were brown lesions of leaves and canker on one to several branches, leaves of the whole tree turned wilted. Cross sections of diseased plants revealed irregularly shaped brown discoloration in the xylem of the trunk. A fungus was consistently isolated from the leaf when diseased leaves were incubated between two slices of fresh carrot root. Spore masses were picked from the apices of perithecia and transferred to malt extract agar medium (MEA) and incubated at 25°C. After perithecium formation, observed perithecia were black, globose (176.0 to 303.2 × 186.0 to 274.3 μm) and showed a long black neck (634.2 to 809.9 μm). Ascospore had the typical format of a "hat" with dimensions of 4.6 to 6.3 × 3.3 to 4.8 μm. Cylindrical endoconidia (7.1 to 36.1 × 2.9 to 6.0 μm) were found. Chlamydospores were ovoid or obpyriform and smooth (8.6 to 12.1 × 6.9 to 12.1 μm). PCR amplification was carried out for one isolate, YT2. The ITS region of rDNA was sequenced using the procedures of Thorpe et al (3). Analysis of ITS sequence data (GenBank Accession Nos. KF963101 and KF963102) showed that the isolates were 99% homologous to the isolate of Ceratocystis fimbriata from diseased Colocasia esculenta in Cuba, China, and Hawaii (AY526304 to 06) by BLAST analysis. Therefore, the fungus was identified as C. fimbriata based on morphological and molecular characteristics. Pathogenicity of the six isolates from this study was tested by inoculation of 1-year-old pot grown seedlings of loquat. The soil of six plants was inoculated by drenching with 40 ml spore suspension (106 spores/ml). Control plants were inoculated with 40 ml of sterile distilled water. The plants were maintained in a controlled greenhouse at 25°C and watered weekly. After inoculation for 1 week, all plants produced wilt symptoms; as the disease progressed, leaves withered and died after 3 weeks of inoculation while control plants remained symptomless. C. fimbriata was successfully re-isolated from the infected trees and no fungal growth was observed in the controls. The pathogenicity assay showed that C. fimbriata was pathogenic to loquat. To our knowledge, this is the first report of C. fimbriata causing wilt of loquat in China. Since C. fimbriata is one of the most aggressive plant pathogens on a wide variety of perennial as well as agronomic crop plants worldwide, and the infection can pose a significant threat to the production of loquat, it is critical to deploy appropriate management strategies to limit the fungus spread.

Non-stationary drying of carrot: Effect on product quality

This paper presents the results of studies concerning the quality of food materials like vegetables subjected to convective drying in non-stationary conditions. The effect of different frequencies and amplitudes of the periodically changeable drying air temperature on the quality of dried carrot (Daucus carota L.) has been investigated. The indicators of the quality such as the color change, the water activity and the retention of β-carotene were analyzed. The slices of carrot root were used as the material for experimental tests. It has been shown that the non-stationary drying carried out in a prescribed variable air temperature significantly minimized the unfavorable effects in color changes, β-carotene degradation, and decay phenomena.

Phenolic metabolism in root slices of selected carrot cultivars

Carrot root slices, stored for 4 days at 20 °C reacted with a strong accumulation of total phenols, especially chlorogenic acid. A significant accumulation of isocoumarin content within the peel was observed in stored slices. Synthesis of phenols was accompanied by an increase in phenylalanine ammonia lyase activity, wound induced respiration and ethylene production.

The corn wound metabolite DIMBOA causes cell death in tobacco and corn

DIMBOA (2,4-dihydroxy-7-methoxy-1,4(2H)-benzoxazin-3-one), a corn (maize) secondary metabolite, is lethal to the cells of tobacco, carrot root slices and to both DIMBOA-producing (B73) and non-producing (bxbx) varieties of corn. Application of DIMBOA to wounded tobacco leaf petioles causes a black lesion and shrivelling of the whole leaf, presumably resulting from death of DIMBOA-treated cells and subsequently uneven growth of the leaf blade. Microscopic examination reveals that DIMBOA treatment causes plasmolysis, cell collapse and disruption of cells. DIMBOA also causes browning and necrosis in the leaf-spot assay on tobacco and on high- and low-DIMBOA varieties of corn. MBOA, a degradation product of DIMBOA, does not cause significant damage to the corn leaf.

The roles of aspartic proteinase and endo-pectin lyase enzymes in the primary stages of infection and pathogenesis of various host tissues by different isolates of Botrytis cinerea Pers ex. Pers

A single spore isolate of Botrytis cinerea, obtained from carrot tissue, produced an aspartic proteinase and an endo-pectin lyase both in vitro in a liquid medium containing 0.4% gelatin as protein source and in infected carrot tissue. Both enzymes caused cell death and induced resistance to B. cinerea in surface cell layers of carrot root slices when applied at relatively low concentrations. At higher concentrations, both caused extensive cell death in carrot tissue and of carrot suspension cells. Neither enzyme lysed osmotically balanced carrot protoplasts unless carrot cell wall preparations were also present. Plasmolysed carrot cells were protected from the phytotoxic activity of the endo-pectin lyase but not from proteinase-induced damage. It is suggested that the phytotoxic activity of both the endo-pectin lyase and aspartic proteinase is indirectly caused by the toxic effect of cell wall components released by each enzyme. Accumulation of 6-methoxymellein was induced in carrot root slices by soluble, non-phytotoxic, elicitors released as a result of the action of aspartic proteinase or endo-pectin lyase on carrot cell walls. Proteinase production was an early event in infection of carrot, strawberry, raspberry, cabbage, grape and broad bean by eight different isolates of the pathogen. The level of infection in these hosts was markedly reduced when spores were pre-treated with pepstatin, a specific inhibitor of the aspartic proteinase, implying a primary role for this enzyme in pathogenesis.

Induction of 6-methoxymellein 6-hydroxymellein production in carrot cells

6-Methoxymellein, a phytoalexin of carrot, and 6-hydroxymellein, its presumed biosynthetic precursor, were isolated from carrot root slices infected with Sclerotium rolfsii and Fusarium solani. Elicitation of both 6-methoxymellein and 6-hydroxymellein synthesis was achieved also in carrot cell suspension cultures by direct addition of pectinolytic enzymes such as Aspergillus niger pectinase highly purified Fusarium moniliforme endo-polygalacturonase (EC. 3.2.1.15). 6-Hydroxymellein was preferentially detected inside the cells while 6-methoxymellein was always found in the culture medium. Changes in pH values at the moment of elicitation of carrot cell suspension cultures influenced the amounts of both phytoalexins synthesized.

Ethylene-induced Respiration in Thin Slices of Carrot Root

Summary Slicing of carrot (Daucus carota L.) root evokes a typical wound-induced respiration with a peak at 18 to 24 hours. While ethylene at 10μ11-1 in air has no effect on the perceived wound-induced rise, it prevents the post-peak respiratory decline and causes a second, more pronounced respiratory climax at 96 hours. Thus the predominant ethylene-induced response can be separated from that elicited by wounding. Oxygen advances the ethylene response so that it is superimposed upon, and extends beyond, the wound-induced climax. When the administration of ethylene in oxygen is delayed 48 hours, however, the ethylene peak is well separated from the wound-induced peak. Stimulation by ethylene depends on its continuous presence; respiration drops immediately on ethylene removal. Actinomycin D totally blocks the development of both the wound- and ethylene-induced respiration. The inhibitor also prevents any decline after the respiratory climax. Cycloheximide stimulates carrot slice respiration, the extent of stimulation being greater in the presence of ethylene. The implications of these effects are discussed and strongly commend the versatile slice system for studies of ethylene action and cycloheximide-induced metabolic stimulation.

Effects of free radical scavengers on 6-methoxymellein accumulation and resistance to Botrytis cinerea in carrot root slices

6-Methoxymellein (6-MM) accumulation in carrot root slices was inhibited by the free radical scavengers salicylhydroxamic acid (SHAM) and propyl gallate (PG). 6-MM accumulation was insensitive to KCN. Linoleic acid and lipoxygenase products of linoleic acid inhibited germination and growth of the carrot pathogen Botrytis cinerea. SHAM and PG induced susceptibility in carrot slices to B. cinerea. A possible role is discussed for fatty-acid peroxy radicals in the mechanism of 6-MM accumulation, and the resistance response in carrot slices to B. cinerea.

Effects of ethylene biosynthesis in carrot root slices on 6‐methoxymellein accumulation and resistance to Botrytis cinerea

The role of ethylene biosynthesis in the resistance response of carrot (Daucus carota L., cv. Chantenay red‐cored) slices to infection by Botrytis cinerea Pers. ex Pers. was investigated using aminoethoxyvinylglycine (AVG) and inhibitor of 1‐aminocyclopropane‐1‐carboxylic acid (ACC) synthase (EC 4.4.1.‐), and norbornadiene, an inhibitor of ethylene binding. Carrot slices became susceptible to a normally non‐invasive level (105 ml‐1) of spores of B. cinerea after treatment with AVG. ACC partially reversed the susceptibility induced by AVG. The ability of a crude pectic enzyme preparation from B. cinerea to induce resistance to a normally invasive level of B. cinerea spores (106 ml‐1) was prevented by AVG. Accumulation of the carrot phyto‐alexin 6‐methoxymellein (6‐MM) was prevented by norbornadiene, but it had no effect on the resistance response. An event associated with ethylene biosynthesis other than 6‐MM accumulation appears to be responsible for the resistance of carrot slices to infection by B. cinerea.

Cell death, 6-methoxymellein accumulation, and induced resistance to Botrytis cinerea in carrot root slices

The carrot phytoalexin 6-methoxymellein (6-MM) accumulated in carrot root slices in response to several treatments which caused cell death. Maximal concentrations (360 μg g −1 fresh weight) 24 h after inoculation, were induced using a noninvasive level of live inoculum of spores of Botrytis cinerea (1 × 10 5 spores ml −1). Optimal freezing times for 6-MM accumulation were between 2 and 10 s (approx. 45 μg g −1 fresh weight at 24 h). Increasing spore concentrations which led to invasion, or lengthening freezing periods, increased the number of cell layers killed below the surface, but resulted in decreased levels of 6-MM accumulation. Ethylene (101 μl l −1) did not cause cell death or induce resistance, although it caused a similar rate of 6-MM accumulation over a 24 h period as partial freezing. It is concluded that cell death is not a necessary prerequisite for 6-MM accumulation.

Inhibition of cyclic nucleotide phosphodiesterase by carrot phytoalexin

The carrot phytoalexin, 6-methoxymellein, was isolated and purified from carrot root slices infected by the fungus Chaetomium globosum. It inhibited the basal and calmodulin-promoted activity of cyclic nucleotide phosphodiesterase. The inhibition of calmodulin-promoted diesterase activity was reduced by increasing the concentration of calmodulin or calcium while the inhibition of basal diesterase activity was reversed by the addition of magnesium to the assay mixture of the enzyme.

The effect of Sa and MiniSa plasmids on the induction of plant crown galls and hairy roots byAgrobacterium tumefaciens andAgrobacterium rhizogenes

The Inc-W group plasmid Sa or its derivative MiniSa were introduced into two strains ofAgrobacterium tumefaciens with Ti plasmids, one strain ofA. tumefaciens with the Ri plasmid and oneA. rhizogenes strain with the Ri plasmid. The effect was similar in allAgrobacterium strains. The pSa suppressed fully the virulence ofAgrobacterium strains (i.e. their ability to induce tumor growths - crown galls or hairj7 roots) inKalanchoe plants and carrot root slices. The MiniSa plasmid caused only a slight decrease of the frequency and size of tumor growths induced. The mechanism of suppression of virulence by the Sa plasmid inAgrobacterium tumefaciens andAgrobacterium rhizogenes seems to be similar.

Elicitation of phytoalexin production in cultured carrot cells

6-Methoxymellein accumulated in cultured carrot cells following exposure to an extract prepared from carrot root slices or cultured carrot cells infected by Chaelomium globosum. The elicitoractive substance in the extract of fungi-infected carrot was heat stable, but susceptible to the treatments with acid, Pronase E, trypsin and pectinase. Elicitor-active substances were also obtained by partial hydrolysis of carrot cells with proteolytic or pectinolytic enzymes, suggesting that the cellular fragments released by these enzymes act as elicitors and stimulate 6-methoxymellein production in carrot cells. The fact that 6-methoxymellein also accumulated in carrot cells after the direct addition of these enzymes to the culture medium support this hypothesis. In contrast, hydrolysates of fungal homogenates showed no significant effect on 6-methoxymellein induction; however, the fungal hydrolysates caused browning in carrot cells indicating that polyphenols rather than 6-methoxymellein were accumulated.

Insolubilization of hydroxyproline-rich cell wall glycoprotein in aerated carrot root slices

The hydroxyproline-rich glycoprotein of plant cell walls is secreted from the cytoplasm as a soluble monomer which slowly becomes insolubilized. A tyrosine derivative, isodityrosine, is formed in the cell wall during this insolubilization and could serve as a protein-protein crosslink. Glycoprotein insolubilization is inhibited by peroxidase inhibitors and free radical scavengers, the most effective of which is L-ascorbate. These data support a hypothesis that the hydroxyproline-rich cell wall glycoprotein forms a covalently crosslinked wall network under the control of an extracellular peroxidase/ascorbate oxidase system.

The Induced Resistance Response of Carrot Root Slices to Heat-killed Conidia and Cell-free Germination Fluid of Botrytis cinerea Pers. ex Pers. 1. The Possible Rôle of Cell Death

The Induced Resistance Response of Carrot Root Slices to Heat-killed Conidia and Cell-free Germination Fluid of Botrytis cinerea Pers. ex Pers. 1. The Possible Rôle of Cell Death

The Induced Resistance Response of Carrot Root Slices to Heat-killed Conidia and Cell-free Germination Fluid of Botrytis cinerea Pers. ex Pers.

The Induced Resistance Response of Carrot Root Slices to Heat-killed Conidia and Cell-free Germination Fluid of Botrytis cinerea Pers. ex Pers.