Rhodosporidium paludigenum induced resistance in Ponkan mandarin against Penicillium digitatum requires ethylene-dependent signaling pathway

This work determined if the ethylene dependent signal pathway was required for antagonist-mediated fruit defense mechanisms through investigation of disease resistance against Penicillium digitatum in Ponkan mandarin induced by 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception, and Rhodosporidium paludigenum. Blocking ethylene perception with 1-MCP resulted in an increase in ACS1, ACS2 and ACO expression, and consequently an increase in ethylene production during mechanical wounding and resistance induction. The expression of the ethylene receptors ETR1, ETR2 and ETR4 as well as ethylene response factor (ERF) were observed with similar responses to yeast and 1-MCP stimuli, with ETR3 mRNA accumulation being the most sensitive to yeast application while ERS1 was the least sensitive. When applied at concentrations greater than 500 nL L⁻¹, 1-MCP pre-fumigation significantly reduced the fruit's natural protection and R. paludigenum induced disease resistance to Penicillium decay, indicating that ethylene perception was involved in inducting disease resistance. Moreover, expression of the defensive genes CHI, β-1,3-glucanase, PAL and CIN up-regulated by yeast was inhibited to different degrees by the 1-MCP pre-treatment. This study provides evidence that the biocontrol yeast R. paludigenum increased disease resistance in Ponkan mandarin against P. digitatum infection due to ethylene and signaling pathway dependent mechanisms.     

Inhibition of green mold disease in mandarins by preventive applications of methyl jasmonate and antagonistic yeast Cryptococcus laurentii

The objective of this study was to evaluate the preventive activity of methyl jasmonate (MeJA) alone and in combination with antagonistic yeast in suppressing green mold decay in citrus fruit, and to explore the mechanisms involved. At 100 μmol/L, MeJA inhibited disease incidence and lesion diameter of mold decay compared with the control (P < 0.05) The preventive application of Cryptococcus laurentii at 1 × 10⁸ cells/mL combined with 100 μmol/L MeJA reduced green mold incidence compared to the control and the other treatment groups (P < 0.05) when tested in wounded citrus fruit inoculated with Penicillium digitatum. MeJA and C. laurentii induced higher activity of polyphenol oxidase, peroxidase and catalase than control. Moreover, treatment with MeJA and C. laurentii induced a rise in the mRNA expression level of PR5 (pathogenesis-related protein family 5), which was stronger than in the single-treatment groups and the control. In addition, 100 μmol/L MeJA improved the rapid proliferation of C. laurentii in citrus fruit wounds. This combined treatment can induce natural resistance and stimulate the proliferation of antagonistic yeast on the fruit surface.     

Use of citral incorporated in postharvest wax of citrus fruit as a botanical fungicide against Penicillium digitatum

The antifungal activity of citral against Penicillium digitatum, the causal agent of citrus green mold, was tested by in vitro and in vivo experiments. In vitro assays showed that the minimum inhibitory concentration and the minimum fungicidal concentration (MFC) were both 4000 μL L⁻¹. Results of in vivo tests demonstrated that wax + citral (1× MFC) treatment did not effectively inhibit the growth of P. digitatum in Ponkan mandarin fruit, whereas wax + citral (10× MFC) treatment significantly decreased the incidence of green mold after 6 days of storage at 25 ± 2 °C. Wax + citral (10× MFC) treatment remarkably increased the content of vitamin C and antioxidant enzyme activities such as catalase, superoxidase dismutase, and peroxidase but decreased the activities of phenylalanine ammonia lyase, polyphenol oxidase, and malonaldehyde. The treatment had minor effects on the pH, coloration index, and total soluble solids. This study provided theoretical data for the practical application of citral on citrus fruit quality during postharvest storage.     

Wound response in orange as a resistance mechanism against Penicillium digitatum (pathogen) and P. expansum (non-host pathogen)

Penicillium digitatum is the most devastating postharvest pathogen of citrus. In addition, Penicillium expansum is the main pathogen of pome fruit, although recent studies have demonstrated its ability to infect oranges under some conditions. In this study, we evaluated wound response in ‘Valencia’ oranges harvested at three different maturity stages and the effect of wound response on the establishment of both pathogens when fruit were stored at two different temperatures (20 and 4 °C). The effect of wounding and pathogen inoculation on lignin content, was also quantified. Lastly, the expression of several phenylpropanoid pathway-related genes was also analyzed by semi-quantitative RT-PCR. Results indicated that, in general, P. digitatum exhibited lower decay incidence and severity as time between wounding and inoculation increased. Decay incidence and severity were higher in fruit from the over-mature harvest than in fruit from immature and commercial harvests. P. expansum was able to infect fruit at 20 °C but lesions were small compared to lesion size of fruit stored at 4 °C. Lignin content in wounded fruit (control) and in samples wounded and inoculated with P. expansum was highest in fruit from the immature harvest at 7 d post-wounding and inoculation. Wounded fruit had higher expression of pal1, comt1 and pox1 genes at 48 h than at 24 h. However, samples inoculated with P. digitatum showed lower expression at 48 h than at 24 h. Our results indicated that maturity and storage temperature play an important role in orange wound response.     

Characterization of sensitivity of grove and packing house isolates of Penicillium digitatum to pyrimethanil

In most northeast Argentinean citrus packing houses, postharvest fungicide treatments are based on the use of thiabendazole and imazalil. However, these fungicides have been used in a manner highly conducive to the selection and proliferation of resistant biotypes of Penicillium digitatum, the main fruit decay fungus in the area. Recently, a new fungicide, pyrimethanil (PYR), was introduced to control molds. Aims of this study were to determine the baseline sensitivities for PYR against isolates of P. digitatum considering its use in the region is not yet widespread and to evaluate the control of the fungus in vivo. One hundred and nine (109) P. digitatum isolates were collected from diseased fruit within citrus groves (43 isolates) and packing houses (66 isolates). EC₅₀ was determined for each isolate by measuring colony diameters on different agar dilutions of the fungicide. The mean EC₅₀ value of the green mold isolates collected from the groves was 0.14 ± 0.03 mg L⁻¹ while the mean EC₅₀ of those collected from packing houses was 0.13 ± 0.05 mg L⁻¹. No resistant isolates were found in the field where the fungicide is not used, while one isolate originated from a packing house showed an EC₅₀ of 3.40 mg L⁻¹, 26-fold higher than the mean level. This isolate was collected from lemons stored in cool rooms of a packing house where PYR had not been used. Fruit decay by sensitive isolates was reduced approximately 80% by PYR applied at 500–600 mg L⁻¹ by immersion for 60 s at room temperature to inoculated oranges and mandarins. In contrast, the resistant isolate was not controlled by PYR applied at 1000 mg L⁻¹. Thus, the introduction of PYR applied into packing houses should be done carefully and control strategies should be implemented in order to minimize the development of resistant isolates.     

Assessment of blue light treatments on citrus postharvest diseases

Exposure of mature ‘Fallglo’ tangerine fruit to blue light with a photon fluence rate 40 μmol m⁻² s⁻¹ reduced symptom development of blue mold (Penicillium italicum), green mold (Penicillium digitatum), and stem end rot (Phomopsis citri) postharvest decays. Direct exposure to blue light was required to reduce decay caused by Penicillium. Blue light (40 μmol m⁻² s⁻¹) reduced in vitro fungal growth of P. italicum and P. citri. The growth of P. digitatum was more tolerant to blue light, however, the activity of fungal polygalacturonase was reduced by blue light at the intensity of 40 μmol m⁻² s⁻¹. Gas chromatography–mass spectrometry analysis identified 29 chemical constituents in flavedo oil; blue light induced only octanal accumulation. Application of octanal suppressed growth of P. italicum, P. digitatum, and P. citri in vitro. Treatment of fruit with octanal at 5 mM or 50 mM suppressed symptom development caused by Penicillium and P. citri, but discolored the peel. Inhibition of postharvest decays by blue light may be due to a combination of inhibition of fungal growth and induction of defensive responses in the host.     

Performance of fogged disinfectants to inactivate conidia of Penicillium digitatum within citrus degreening rooms

Fogging with formaldehyde of citrus packinghouses when the fruit are absent is a practice to control conidia of Penicillium digitatum (Pers.) Sacc., the cause of citrus green mold. Replacements for formaldehyde in these facilities are needed because of worker and environmental health issues. To evaluate the effectiveness of candidate sanitizers, craft wood sticks with conidia of P. digitatum were attached throughout commercial citrus ethylene degreening rooms and either water alone or the sanitizers were applied. The rooms were 20 ± 2 °C and humidified to 85–90% relative humidity. Aldehydes, peroxygen compounds, sodium hypochlorite, chlorine dioxide, quaternary ammonium, alcohols, one phenolic compound, and one organic acid were applied with a compressed air assisted atomizer or fan atomizer in a volume of approximately 6 L per 100 m³ of room volume dispensed over a 3 h period. Rates applied were expressed as active ingredient per m³ of room volume. All were compared to formaldehyde applied at 1.98 g m⁻³ of room volume. After 24 h, the craft wood sticks were retrieved, and germination of the conidia assessed. Five sanitizers reduced germination of conidia by more than 95% and equaled formaldehyde in effectiveness. They were (effective rates): (1) glutaraldehyde (0.1 g m⁻³); (2) hydrogen peroxide (4.4 g m⁻³); (3) Citrisol (1.0 g m⁻³), a proprietary mineral oxychloride oxidizer; (4) acetic acid (5.3 g m⁻³); and (5) peracetic acid (2.4 g m⁻³). The toxicity of effective sanitizers was determined by exposure of P. digitatum conidia for 10 min to concentrations of each and the proportion of survivors used to estimate EC₅₀ and EC₉₉ concentrations. The toxicity of the sanitizers in this assay did not predict their effectiveness when applied by fogging, probably because other factors, such as distribution, persistence, droplet size, or vapor pressure also influenced their effectiveness.     

Effect of ethylene degreening on the development of postharvest penicillium molds and fruit quality of early season citrus fruit

The effect of commercial degreening with ethylene gas on fruit susceptibility and quality and development of postharvest green (GM) and blue (BM) molds on early season citrus fruit was investigated. Each cultivar was harvested with different peel color indexes (CI). Fruit were exposed for 3 d to 2 μL L⁻¹ ethylene at 21 °C and 95–100% RH before or after artificial inoculation with Penicillium digitatum or Penicillium italicum. Control fruit were kept at the same environmental conditions without ethylene. Fruit were stored at either 20 °C for 7 d or 5 °C for 14 d and disease incidence (%) and severity (lesion diameter) were assessed. No significant effect of commercial degreening was observed on fruit susceptibility to both GM and BM on citrus cultivars inoculated after degreening. Likewise, no significant effect was observed on disease incidence on citrus cultivars inoculated before degreening and stored at either 20 °C for 7 d or 5 °C for 14 d. In contrast, in cultivars like ‘Clemenules’ mandarins and ‘Navelina’ oranges, degreening significantly increased the severity on fruit with higher initial CI (−3.6 and 1.7, respectively). GM and BM severity on degreened and control ‘Clemenules’ mandarins incubated at 20 °C for 7 d was 146 and 118 mm and 56 and 46 mm, respectively. In general, commercial degreening did not significantly affect external and internal quality attributes of citrus cultivars. Commercial degreening after inoculation of less green (more mature) fruit showed a trend to increase mold severity, presumably through an aging effect (acceleration of peel senescence).     

Development and application of a SCAR marker to monitor and quantify populations of the postharvest biocontrol agent Pantoea agglomerans CPA-2

Pantoea agglomerans CPA-2 is an effective biocontrol agent of postharvest diseases of citrus and pome fruit. A monitoring technique was developed for its identification and to quantify its populations. The methodology used consisted of (i) searching for a semi-selective medium, (ii) identification of molecular markers and (iii) monitoring population dynamics in a commercial trial. As a semi-selective medium, Malonate Broth Agar supplemented with tetracycline hydroxychloride and incubation at high temperature (max. of 40 °C) facilitated the selective recovery of P. agglomerans CPA-2 colonies. The RAPD technique was applied to a collection of 13 strains of P. agglomerans, including CPA-2. Among the 12 primers tested, OPL-11 amplified a fragment (about 720 bp) specific to strain CPA-2. On the basis of this fragment, two SCAR markers were amplified using a primer pair derived from OPL-11 elongation. A first SCAR marker of 720 bp was specifically amplified for the strain CPA-2 and a second one of 270 bp was obtained for all P. agglomerans strains tested, including CPA-2. Commercial trials demonstrated a significant reduction of decay with the treatment of formulated cells of P. agglomerans CPA-2. Population dynamics of CPA-2 in commercial trials were determined on fruit surfaces and in the environment using both the classical plating technique and PCR with SCAR primers. In general, no significant differences were observed between results obtained from the two methods. On fruit surfaces, 1 day after CPA-2 applied its population by classical methods was 4.37 × 10⁶ cfu wound⁻¹ and at the end of the experiment the population increased to 5.8 × 10⁵ cfu wound⁻¹. The percentages of colonies identified as P. agglomerans CPA-2 at these sampling times using SCAR primers were 90 and 95%, respectively. Population dynamics in the environment to evaluate the environmental fate of P. agglomerans CPA-2 showed that it has a limited persistence and limited capacity for dispersion.     

Imazalil residue loading and green mould control on citrus fruit as affected by formulation,solution pH and exposure time in aqueous dip treatments

Green mould, caused by Penicillium digitatum, is responsible for major postharvest fruit losses on the South African fresh citrus export market. Some of these losses as well as fungicide resistance development can be attributed to sub-optimal imazalil (IMZ) residue loading on citrus fruit (<2 μg g⁻¹), which is commonly the case in South African packhouses. This will result in loss of control and sporulation inhibition on decayed fruit. IMZ formulation [IMZ sulphate and emulsifiable concentrate (EC)], solution pH (IMZ sulphate at 500 μg mL⁻¹ buffered with NaHCO₃ or NaOH to pH 6 and 8) and exposure time (15–540 s) were investigated in order to improve IMZ residue loading and the green mould control on Clementine mandarin, ‘Eureka’ lemon, and navel and Valencia orange fruit. Exposure time had no significant effect on residue loading in the unbuffered IMZ sulphate solution (pH 3). No differences were observed between the pH buffers used, but residue loading improved with increase in pH. The maximum residue limit (MRL) of 5.0 μg g⁻¹ was exceeded following dip treatment in the IMZ EC (after 75 s exposure time), and IMZ sulphate at pH 8 using NaHCO₃ (77 s) or NaOH (89 s) as buffer. The MRL was exceeded after 161 s in IMZ sulphate solutions buffered at pH 6 with either NaHCO₃ or NaOH. An IMZ residue-loading curve was prepared from which residue levels can be predicted for the control of IMZ-sensitive and IMZ-resistant isolates of P. digitatum. From this model the benchmark residue level for 95% control of an IMZ-sensitive isolate and of an IMZ-resistant isolate were predicted to be 0.81 and 2.64 μg g⁻¹, respectively. Residue loading can be improved by adjusting the pH level of an IMZ sulphate solution to 6 or by using the IMZ EC formulation, but exposure time should be restricted to 45 s so as not to exceed the MRL. Conversely, sufficient exposure time of ≈90 s in an unbuffered IMZ sulphate solution (pH 3) will result to improved green mould control, but with residue loading below 2 μg g⁻¹. The resistant isolate could not be controlled adequately with residue levels below the MRL, therewith indicating the practical relevance of IMZ resistance.     

Hot water dipping treatments on Tarocco orange fruit and their effects on peel essential oil

The most common and serious diseases which affect citrus fruit after harvest in Italy are induced by Penicillium digitatum Sacc. and Penicillium italicum Weh., responsible respectively for green and blue mold rots. This paper deals with the effectiveness of hot water dipping (HWD) treatments as alternative means to control postharvest decay on Tarocco orange fruit [Citrus sinensis (L.) Osbeck], and their effect on fruit quality with special regard to peel essential oils. Selected treatments were HWD at 52 °C for 180 s and at 56 °C for 20 s. These treatments were compared with an effective fungicide standard treatment (Imazalil) and an untreated control. The results showed that HWD at 56 °C for 20 s was more effective in inhibiting P. digitatum spore germination than HWD at 52 °C for longer exposure time. In addition, HWD treatment at 56 °C significantly increased the level of alcohols, esters and aliphatic (fatty) aldehydes. Therefore, the lowest values of decay incidence recorded in HWD fruit treated at 56 °C may be due to the increase in oxygenated monoterpenes, esters and aldehydes. Finally, HWD treatments did not cause surface damage or color change and did not influence internal quality parameters.     

Induction of H2O2-metabolizing enzymes and total protein synthesis by antagonistic yeast and salicylic acid in harvested sweet cherry fruit

The immersion of sweet cherry fruit in Pichia membranefaciens at a concentration of 5 × 10⁷ cells ml⁻¹ or in salicyclic acid (SA) at 0.5 mM for 10 min reduced the incidence of decay and lesion size caused by Penicillium expansum. Without pathogen inoculation, peroxidase (POD) activity was enhanced in yeast-treated fruit, but activities of catalase (CAT) and superoxide dismutase (SOD) showed a decrease in the same fruit. SA-treatment significantly inhibited CAT activity, but stimulated SOD and POD activities. After inoculation with P. expansum, CAT activity decreased and SOD activity increased in both yeast- and SA-treated fruit. No obvious difference was found in POD activity between treatments and water control. Treatments with yeast and SA changed the expression of POD isozymes. In addition, yeast and SA treatment increased total protein content of sweet cherry and up-regulated 33 and 47 kDa protein bands shown by SDS-PAGE. These results indicated that yeast- and SA-treatments induced synthesis of anti-oxidant enzymes and specific proteins, which may play a role in the resistance against postharvest blue mold.     

Effect of chitin on the antagonistic activity of Rhodosporidium paludigenum against Penicillium expansum in apple fruit

This study aimed to investigate the effect of chitin on the antagonistic activity of Rhodosporidium paludigenum against Penicillium expansum, the cause of blue mold in apple fruit, and the possible mechanisms involved. Our results showed that biocontrol efficacy and population growth of R. paludigenum were greatly enhanced when it was harvested from nutrient yeast dextrose with added chitin (NYCB) medium compared with that harvested from nutrient yeast dextrose (NYDB) medium. The ability of R. paludigenum produced in NYCB to induce resistance to blue mold in apple fruit was significantly enhanced. The enhanced disease control efficacy was correlated with higher levels of polyphenoloxidase (PPO) and superoxide dismutase (SOD) activities in apple fruit treated with R. paludigenum. Moreover, the SOD and catalase (CAT) activities of R. paludigenum were stimulated by cultivating in NYCB, while malondialdehyde (MDA) accumulation in the yeast cells was suppressed. These results indicated that adding chitin to normal media might be an effective method to improve the antagonistic activity of R. paludigenum and the active oxygen metabolism of R. paludigenum might be closely related to the biocontrol activity of the yeast.     

Real time polymerase chain reaction for rapid and quantitative determination of Cystofilobasidium infirmominiatum on the surfaces of apple,pear, and sweet cherry fruit

The objectives of this study were to develop primers and a real time PCR protocol for the postharvest biocontrol yeast Cystofilobasidium infirmominiatum (Cim). The application of this technology was developed to quantify Cim on the surfaces of apple, two pear cultivars, and sweet cherry fruit treated over a range of concentrations. Statistically significant relationships were observed between Cim DNA on fruit surfaces, expressed as μg/m², and CFU/L of dip suspensions for apple, pear, and sweet cherry. In addition, the relationship for each fruit was significantly different from the other three fruits. Threshold values of concentrations of Cim DNA on the fruit surface were calculated based on regression equations and a dose of 2.0 × 10¹¹ CFU/L of dip suspension, the dose for optimum decay control, and were 4.8, 7.0, 16.5, and 25.2 μg/m² for Bosc pear, Lapins sweet cherry, d’Anjou pear, and Golden Delicious apple, respectively. Monitoring Cim DNA concentration on fruit surfaces will assure that Cim is being properly applied to fruit and that a sufficient number of cells are present for optimum decay control.     

Superoxide anion and hydrogen peroxide in the yeast antagonist–fruit interaction: A new role for reactive oxygen species in postharvest biocontrol?

The importance of reactive oxygen species (ROS) in plant defense responses against certain pathogens is well documented. There is some evidence that microbial biocontrol agents also induce a transient production of ROS in a host plant which triggers local and systemic defense responses to pathogens. The ability of biocontrol agents used to control postharvest diseases to induce defense-related oxidative responses in fruits, however, has not been explored. Here we show that the yeast antagonists, Metschnikowia fructicola (strain 277) and Candida oleophila (strain 182) generate greater levels of super oxide anion (O₂⁻) on intact fruit surfaces (poor in nutrients) then those applied on a nutrient-poor agar medium. Even though yeast antagonists show a high level of O₂⁻ on nutrient-rich media, when applied on fruits around wounds (areas abundant in nutrients) accumulation of O₂⁻, as detected by nitro blue tetrazolium staining, occurred much more rapidly on the latter. Using laser scanning confocal microscopy we observed that the application of M. fructicola and C. oleophila into citrus and apple fruit wounds correlated with an increase in H₂O₂ accumulation in host tissue. In citrus fruit, the level of H₂O₂ around inoculated wounds increased by 4-fold compared to controls (wounds inoculated with water) as early as 18 h after inoculation. Yeast continued to stimulate H₂O₂ production in citrus fruit up to 66 h after inoculation and H₂O₂ levels were still 3-fold above the control. Living yeast cells were detected in fruit wounds at this time point indicating the ability of M. fructicola to tolerate host ROS, which has been reported to be an intrinsic characteristic of efficient yeast antagonists. Similar increase in H₂O₂ accumulation around yeast-inoculated wounds was observed in apple fruit exocarp. The present data, together with our earlier discovery of the importance of H₂O₂ production in the defense response of citrus flavedo to postharvest pathogens, indicate that the yeast-induced oxidative response in fruit exocarp may be associated with the ability of specific yeast species to serve as biocontrol agents for the management of postharvest diseases.     

Effects of X-ray irradiation and sodium carbonate treatments on postharvest Penicillium decay and quality attributes of clementine mandarins

The integration of sodium carbonate (SC; dips at 20 °C for 150 s in aqueous 3% SC solutions) treatments and X-ray irradiation (at doses of 510 and 875 Gy) was evaluated on artificially inoculated ‘Clemenules’ clementine mandarins for the control of postharvest green and blue molds, caused by Penicillium digitatum and Penicillium italicum, respectively. Although significant, the reduction of both disease incidence (number of infected fruit) and severity (lesion diameter) on fruit either incubated at 20 °C for 7 days or cold-stored at 5 °C for 21 days was not sufficient for satisfactory disease control under hypothetical commercial conditions. Therefore, the combined treatments could not be a substitute for conventional chemical fungicides. However, pathogen sporulation was greatly inhibited on infected clementines, thus X-irradiation could be of value for management of Penicillium resistant strains and to reduce inoculum levels in citrus packinghouses. X-ray irradiation at 195, 395, 510, and 875 Gy did not influence either decay incidence or the area under the disease progress curve (AUDPC) of lesions of green and blue molds on mandarins inoculated with the pathogens 2, 3, or 6 days after irradiation and incubated for 7 days at 20 °C. Therefore, X-ray treatment did not induce disease resistance in the rind of irradiated fruit. Although X-irradiation at doses up to 875 Gy followed by either 14 days at 20 °C or 60 days at 5 °C caused very slight rind pitting, minor decreases in fruit firmness, and modest increases in juice acetaldehyde and ethanol contents, these changes had no practical impact on fruit quality. Rind color, titratable acidity, soluble solids concentration, maturity index and juice yield were not influenced by irradiation. ‘Clemenules’ can be considered as a clementine cultivar highly tolerant to X-irradiation.     

Phomopsis longanae Chi-induced pericarp browning and disease development of harvested longan fruit in association with energy status

The effects of Phomopsis longanae Chi infection on browning development and disease incidence in relation to energy status in pericarp of harvested longan fruit were investigated. Longan fruit were inoculated for 5 min with P. longanae at 10⁴ spores mL⁻¹, while fruit dipped in sterile deionized water were used as control. These fruits were stored at (28 ± 1) °C and 90% relative humidity for up to five days. The results showed that the browning index, disease incidence, cellular membrane permeability and AMP content increased but the contents of ATP and ADP, and energy charge decreased in pericarp of longan fruit infected by P. longanae. It was suggested that P. longanae infection caused energy deficiency in longan fruit, possibly resulting in accelerated senescence and decreased resistance to pathogen, and thus promoted browning development and disease occurrence.     

Degreening behavior in ‘Fallglo’ and ‘Lee × Orlando’ is correlated with differential expression of ethylene signaling and biosynthesis genes

Two citrus types (‘Fallglo’ and ‘Lee × Orlando’) exhibiting differential fruit degreening response when treated with ethylene were selected. Fruit were harvested at commercial maturity but at different developmental periods (Harvest I, II and III). Rate of color change was greater in ‘Fallglo’ than in ‘Lee × Orlando’ when fruit were treated with 5 μL L⁻¹ of ethylene for 24 h. After 24 h of transfer of fruit to ethylene-free storage, rate of change decreased in ‘Fallgo’ and exhibited varied response in ‘Lee × Orlando’ depending on harvest date. ‘Fallglo’ fruit from Harvests I and II were completely degreened at the end of storage for 7 d; however ‘Lee × Orlando’ were not and were green in color. No difference in seedling triple response was observed between ‘Fallglo’ and ‘Lee × Orlando’ and sequences of the four ethylene receptors were identical between them. Expression of genes involved in ethylene biosynthesis and signaling pathways were studied in flavedo to test if differences in these pathways were correlated with differential ethylene sensitivity of the citrus types. Basal levels of ACS2 and ACO expressions declined as maturity progressed, and ethylene-induced expression of ACS1 and ACO were influenced by fruit maturity. At Harvests I and II, ethylene-induced increase in ACS1 and ACO expressions and ACC levels were greater in ‘Fallglo’ than in ‘Lee × Orlando’. Ethylene treatment influenced MACC content only during Harvest I in ‘Lee × Orlando’. MACC levels were generally higher in ‘Lee × Orlando’ than in ‘Fallglo’. Expressions of ETR1 and ETR2 were ethylene responsive in ‘Fallglo’ and only ETR1 expression was ethylene responsive in ‘Lee × Orlando’. Ethylene had more impact on ETR1 expression in ‘Fallglo’ than in ‘Lee × Orlando’. Ethylene had a negative effect on ETR3 expression which was more pronounced in ‘Lee × Orlando’ than in ‘Fallglo’. Expressions of ERS1, CTR1, EIN2, EIL1 and EIL2 were not affected by ethylene in both citrus types. Expression of chlorophyllase gene and rate of total chlorophyll degradation were higher in ‘Fallglo’ than in ‘Lee × Orlando’ during ethylene treatment. Differential degreening behavior of ‘Fallglo’ and ‘Lee × Orlando’ correlated with peel maturity, and factor(s) downstream of ethylene signaling but upstream of ethylene biosynthesis play a role in the differential sensitivity.     

Combination of hot water,Bacillus amyloliquefaciens HF-01 and sodium bicarbonate treatments to control postharvest decay of mandarin fruit

An antagonistic isolate Bacillus amyloliquefaciens HF-01, sodium bicarbonate (SBC) and hot water treatment (HW) were investigated individually and in combination against green and blue mold and sour rot caused by Penicillium digitatum, P. italicum and Geotrichum citri-aurantii respectively, in mandarin fruit. Populations of antagonists were stable in the presence of 1% or 2% SBC treatment, and spore germination of pathogens in potato dextrose broth was greatly controlled by the hot water treatment of 45 °C for 2 min. Individual application of sodium bicarbonate at low rates and hot water treatment, although reducing disease incidence after 8 weeks or 4 weeks of storage at 6 °C or 25 °C respectively, was not as effective as the fungicide treatment. The treatment comprising B. amyloliquefaciens combined with 2% SBC or/and HW (45 °C for 2 min) was as effective as the fungicide treatment and reduced decay to less than 80% compared to the control. B. amyloliquefaciens HF-01 alone or in combination with 2% SBC or/and HW significantly reduced postharvest decay without impairing fruit quality after storage at 25 °C for 4 weeks or at 6 °C for 8 weeks. These results suggest that the combination of B. amyloliquefaciens HF-01, SBC and HW could be a promising method for the control of postharvest decay on citrus while maintaining fruit quality after harvest.     

Control of green and blue mould on orange fruit by Serratia plymuthica strains IC14 and IC1270 and putative modes of action

Two biological control agents of Serratia plymuthica, strains IC1270 and IC14 applied separately and in combination were evaluated for suppressing Penicillium digitatum (green mould) or Penicillium italicum (blue mould) on orange. These bacteria were effective and controlled both pathogens at 1 × 10⁸ cells/mL. Disease suppression was increased when both bacterial strains were combined. Possible modes of action studied in this work were antibiosis, chitinolytic activity and competition for nutrients. Two mutants of strain IC1270, one deficient in chitinolytic activity and the second deficient in pyrrolnitrin production were obtained by the gene replacement technique. On orange fruit, mutant IC1270-C7 deficient in chitinase production and mutant IC1270-P1 deficient in pyrrolnitrin production showed a similar efficiency against P. digitatum to the parental strain. However, in vitro IC1270-P1 lost its antifungal activity and no inhibition zone was observed when it was tested against P. digitatum or P. italicum. In similar experiments, a chitinase-deficient mutant of strain IC14 was as effective as the parental strain IC14, suggesting no evidence for a possible role of chitinases in controlling green mould caused by P. digitatum. Interactions between strains IC1270 or IC14 and P. digitatum were studied in tissue culture plates with diluted orange peel extract as the nutrient source. Strain IC1270 decreased germination of P. digitatum conidia when it was physically separated from the pathogen by a membrane filter, which permits nutrient and metabolite interchange, while strain IC14 did not affect germination. Significant inhibition of conidial germination of P. digitatum was achieved, however, when the pathogen and IC14 were in physical contact. Competition for nutrients appears to be the main mode of action of strain IC1270, while a direct cell-to-cell interaction between IC14 and the pathogen is needed for antagonism.