Citrus Packinghouses Research Articles

Identification of Geotrichum citri-aurantii and G. candidum in citrus packinghouses in California, their sensitivity to cyproconazole, and incomplete cross-resistance to propiconazole.

High levels of sour rot on propiconazole-treated lemon fruit that were stored for extended times in some California packinghouses in 2020 and 2021 initiated surveys on fungicide sensitivity of the causal pathogen. In isolations from diseased fruit in 2020 to 2023, 157 isolates of Geotrichum spp. were obtained. Using species-specific primers, 143 were determined to be G. citri-aurantii and 15 were G. candidum. Isolates of G. citri-aurantii were either sensitive (EC50 0.06 to 0.34 μg/ml), moderately resistant (EC50 1.20 to 2.34 μg/ml), or highly resistant (EC50 >17.68 μg/ml) to propiconazole. There was incomplete cross resistance to cyproconazole, another demethylation inhibitor fungicide pending postharvest registration on citrus in the United States: isolates sensitive to propiconazole were sensitive, isolates moderately resistant to propiconazole were sensitive, and isolates highly resistant were moderately resistant to cyproconazole (EC50 0.11 to 0.63 μg/ml, 0.19 to 0.73 μg/ml, and 2.66 to 6.79 μg/ml, respectively). All except one isolate of G. candidum were highly resistant to both fungicides (EC50 >9.55). Isolates of both species were all considered sensitive to natamycin (EC50 1.18 to 5.01 μg/ml). In lemon fruit inoculations with G. candidum, the incidence of typical sour rot increased from 4.7% to 68.2% when inoculum was amended 2 µg/ml or 100 µg/ml cycloheximide, respectively, a compound known to suppress host defenses. In co-inoculations with 1:1 mixtures of the two Geotrichum spp., G. candidum was only recovered from the centers of decay lesions, whereas G. citri-aurantii was also obtained from the advancing margins. We conclude that G. candidum is a secondary pathogen of lemons, and its presence was favored by extended late-season storage of senescent fruit with reduced defense mechanisms.

Effects of packinghouse operations on the flavor of ‘Orri’ mandarins

Mandarins have a delicate flavor and are easy to peel and easy to consume. However, they are relatively perishable and suffer from flavor deterioration after harvest. The goal of the current study was to examine the effects of commercial packinghouse operations on the flavor of ‘Orri’ mandarins. For that purpose, we collected fruit from four different points along a commercial citrus packing line: (1) directly from the harvest bin, (2) after application of a hot (53°C) fungicide treatment for 30 s, (3) after waxing, and (4) after waxing and after the fruit had passed through a hot‐air drying tunnel (37°C) for 2 min. The collected fruit were stored for 3 or 6 weeks at 5°C and then kept for five more days under shelf‐life conditions at 22°C. The observed results indicate that the hot fungicide treatment had no effect on flavor quality. However, the waxing and waxing +drying treatments resulted in significant increases in ethanol accumulation, lower flavor‐acceptability scores, and increased off‐flavors. Gas‐chromatography mass‐spectrometry (GC–MS) analysis revealed that the waxing and waxing +drying treatments resulted in particular increases in the levels of alcohol and ethyl ester volatiles; whereas levels of other aroma volatiles (i.e., esters, aldehydes, monoterpenes, and sesquiterpenes) decreased after storage in all fruit samples. Overall, the waxing process in commercial citrus packinghouses increased ethanol and ethyl ester volatile levels and harmed flavor acceptability. These findings demonstrate the need to identify new wax formulations that do not hamper fruit‐flavor quality.

Organic Acid Sanitizers for Natamycin and Other Fungicides in Recirculating Application Systems for Citrus Postharvest Decay Management.

Natamycin is a new postharvest biofungicide for citrus and some other fruit crops in the United States that can be effectively used in recycling drench or flooder treatments. These applications necessitate sanitation of the fungicide solution to ensure that it remains free from contamination by bacteria that are potentially human pathogens. During invitro experiments, heated (48°C) citric acid (1,100 or 2,200 μg/ml) amended with sodium dodecylbenzenesulfonate (SDBS) (60 or 120 μg/ml, respectively) significantly reduced the viability of a nonpathogenic strain of Escherichia coli in natamycin solutions by >5 log10 compared with the control. During laboratory studies with Penicillium digitatum-inoculated lemon fruit, 1,000 μg/ml of natamycin mixed with 1,000 μg/ml of lactic acid or citric acid and with or without SDBS (55 μg/ml) effectively and significantly reduced green mold. Natamycin mixed with lactic acid at ≥2,000 μg/ml, however, caused fruit injury, resulting in browning and rind pitting. Natamycin was incompatible with peroxyacetic acid, resulting in reduced efficacy against green mold. Sodium hypochlorite mixed with natamycin lost its toxicity to E. coli; however, the performance of natamycin was not affected. With heated (average 49°C) drench treatments on an experimental packing line, natamycin (1,000 μg/ml), fludioxonil (300 μg/ml), or azoxystrobin (300 μg/ml) mixed with citric acid (1,000 μg/ml) and SDBS (55 μg/ml) were effective against green mold without fruit injury. At a pH between 3.6 and 3.8, citric acid-SDBS significantly reduced the viability of E. coli by approximately 4 log10 in mixtures with fludioxonil or azoxystrobin, but not with natamycin. However, natamycin at 1,000 μg/ml mixed with 2,000 μg/ml of citric acid and SDBS (55 μg/ml) significantly reduced E. coli counts by >4 log10 within 4 min when the pH was maintained between 3.0 and 3.3, and the efficacy of the fungicide was retained. The use of citric acid with a surfactant can be a viable alternative sanitation method for natamycin in citrus packinghouses utilizing heated recirculating fungicide systems.

First Report of Penicillium ulaiense Causing Postharvest Whisker Mold of Oranges (Citrus sinensis) in Spain

In a survey of postharvest losses in citrus packinghouses located in Valencia province (Spain) conducted during the 2017 to 2018 citrus season, mold symptoms distinctive from those of the most frequent and well-known citrus green and blue molds (caused by Penicillium digitatum [Pers.] Sacc. and Penicillium italicum Wehmer, respectively) were observed in about 1 to 2% of the sampled molded orange fruits (Citrus sinensis L.). Distinctive symptoms on sporulated soft lesions were the grayish blue color of the conidial masses and the presence of whisker-like coremia with tall whitish stalks. This whisker mold was often found in mixed infections with green and/or blue molds in the same fruit. The potential causal agent (isolate IVIA NAV-1) was transferred to potato dextrose agar (PDA) plates and purified by consecutive replating after 7 days of incubation at 25°C. At this time, colonies were mostly circular, compact, 30 to 40 mm in diameter, with dense velvety grayish mycelium and abundant bluish conidia. Conidiophores were sinoid, mostly terverticillate, and conidia were cylindrical to ellipsoidal, smooth, and thin walled, measuring 4.8 to 8.4 × 2.0 to 3.2 μm. Based on these morphological characteristics, the isolate IVIA NAV-1 was tentatively identified as Penicillium ulaiense H.M. Hsieh, H.J. Su & Tzean (Frisvad and Samson 2004). To confirm the identity, molecular identification was performed in a specialized laboratory. The rDNA internal transcribed spacer regions ITS 1 and ITS 2 and the β-tubulin gene were amplified, sequenced, and deposited in GenBank (accessions nos. MH477872 and MH477873, respectively). A BLAST search for ITS showed 100% identity and 100% query coverage with P. ulaiense strain CBS 210.92 (accession no. KC411695.1). A BLAST search for the β-tubulin gene showed 99% identity and 100% query coverage with P. ulaiense isolate 1399 (accession no. LT629297.1). Selected healthy oranges of cultivar Valencia were surface disinfected by dipping in 0.5% sodium hypochlorite for 2 min followed by thorough rinsing in deionized water. Pathogenicity was tested by pipetting 30 µl of a spore suspension (1 × 10⁶ conidia/ml), prepared from 7- to 10-day-old PDA cultures, onto fresh rind wounds, which were made on the equatorial area of disinfected fruit using a sterile, stainless steel rod with a probe tip of 1 mm in width × 2 mm in length (one wound per fruit, total of 20 oranges). Additionally, 10 disinfected, wounded, noninoculated oranges were used as controls. The same procedure was repeated 15 days later with another lot of Valencia oranges. In both tests, disease symptoms were observed on all inoculated fruit after 7 days of incubation at 25°C, and P. ulaiense was consistently reisolated, thereby fulfilling Koch’s postulates. No decay was observed on any of the control fruit. To our knowledge, this is the first report of P. ulaiense causing whisker mold of citrus fruit in Spain. The disease has been previously reported in important citrus-producing areas such as California (Holmes et al. 1993), Egypt (Youssef et al. 2010), Pakistan (Khan et al. 2017), and Korea (Park et al. 2018).

Non-destructive recognition and classification of citrus fruit blemishes based on ant colony optimized spectral information

Fast and accurate assessment of citrus fruit blemishes is critical to improve fruit quality and company profitability of citrus packinghouses and juice processing plants. This study aimed to identify spectral signatures of healthy fruit, and fruit exhibiting symptoms or damage from Huanglongbing (HLB), melanose, oleocellosis (oil spot), wind scar, leafminer and rust mites. Fruit samples were classified using identified spectral information. The current work proposes a characteristic waveband selection method based on the combination of the ant colony optimization (ACO) algorithm and variable selection principles. Six characteristic wavebands for each type of citrus blemishes were determined. Two different classification methods were established by the acquired characteristic wavebands, including simple layer support vector machine (SVM) classification models and tree-type SVM models. After using the tree-type SVM models, classification accuracies of healthy, HLB, melanose, oil spot, wind scar, leafminer and rust mite categories were 98.4%, 90.8%, 95.2%, 92.0%, 90.8%, 95.2% and 96.8%, respectively. The proposed characteristic wavebands selection methods were therefore very effective in extracting features of citrus fruit with these blemishes and the tree-type SVM classification models made it possible to correctly classify the fruit with high detection accuracies and universality.

Mucor Rot-An Emerging Postharvest Disease of Mandarin Fruit Caused by Mucor piriformis and other Mucor spp. in California.

In recent years, an emerging, undescribed postharvest disease was observed on mandarin fruit after extended storage in California. We collected decayed mandarin fruit from three citrus packinghouses in the Central Valley of California in 2015 and identified this disease as Mucor rot caused by Mucor spp. Mucor rot occurred in 11 of the 15 grower lots sampled, and the percentage of Mucor rot in the total decayed fruit varied among affected grower lots, ranging from 3.3 to 93.1% with an average of 49.2%. In total, 197 isolates of Mucor spp. were obtained from decayed mandarin fruit and identified based on internal transcribed spacer sequence and morphological characteristics. Of the 197 isolates, 182 (92.4%) were identified as Mucor piriformis, 7 (3.6%) were M. circinelloides (6 M. circinelloides f. lusitanicus and 1 M. circinelloides f. circinelloides), 4 (2%) were M. racemosus f. racemosus, 3 (1.5%) were M. hiemalis, and 1 (0.5%) was M. mucedo. All species grew at 0 and 5°C, except M. circinelloides, which did not grow at 0°C. Mycelial growth was arrested at 27°C for M. piriformis; 35°C for M. racemosus f. racemosus, M. circinelloides f. lusitanicus, M. hiemalis and M. mucedo; and 37°C for M. circinelloides f. circinelloides. Optimal mycelial growth occurred at 20°C for M. piriformis and M. mucedo, 25°C for M. racemosus f. racemosus and M. hiemalis, 27°C for M. circinelloides f. lusitanicus, and 30°C for M. circinelloides f. circinelloides. M. piriformis grew significantly faster than the other four species at 5 and 20°C, and M. mucedo was the slowest in growth among the five species. Sporangiospores of M. piriformis, M. racemosus f. racemosus, and M. hiemalis germinated at both 5 and 20°C. M. circinelloides germinated at 20°C but did not germinate at 5°C after incubation for 48 h. All five Mucor spp. caused decay on mandarin fruit inoculated with the fungi, and the lesion size caused by M. piriformis was significantly larger than that caused by other species at both 5 and 20°C. Our results indicated that Mucor rot in mandarin fruit in California is caused by Mucor spp. consisting of M. piriformis, M. circinelloides, M. racemosus f. racemosus, M. hiemalis, and M. mucedo, with M. piriformis being the dominant and most virulent species. Previously, M. racemosus was reported on citrus. This is the first report of Mucor rot in citrus caused by M. piriformis, M. circinelloides, M. hiemalis, and M. mucedo.

Occurrence of Fludioxonil Resistance in Penicillium digitatum from Citrus in California

Penicillium digitatum is the causal agent of green mold, one of the most important postharvest diseases of citrus (Citrus spp.). Fludioxonil can be used alone or in combination with azoxystrobin for the control of green mold and other postharvest diseases on citrus. Baseline sensitivity to fludioxonil in P. digitatum populations from California citrus packinghouses has been previously established (Kanetis et al. 2008). To monitor resistance to fludioxonil in P. digitatum, 20 Penicillium spp. isolates were obtained from decayed oranges by isolation from decayed tissue and 22 isolates were recovered from potato dextrose agar plates amended with 0.5 mg/liter fludioxonil (Kanetis et al. 2006) and exposed to packinghouse air for 5 min each sampling time during March to July 2013 in two California citrus packinghouses. All isolates were single-spore cultured and identified as P. digitatum based on morphological characters (Pitt 1979). The sequence analysis of the internal transcribed spacer (ITS) region, using the primers ITS1/ITS4, was conducted to confirm the species-level identification. A MegaBLAST search showed that the sequences of all isolates had 99% homology (E-value = 0.0) with that of P. digitatum deposited at GenBank (Accession No. AF033471.1). In a mycelial growth assay, 15 of the 42 P. digitatum isolates were able to grow at 0.5 μg/ml, a previously established discriminatory concentration (Kanetic et al. 2006). EC50 values (the effective concentration that inhibits fungal growth by 50% relative to the control) of fludioxonil for the resistant isolates were >100 mg/liter following a method described by Li and Xiao (2008). To assess whether fludioxonil at the label rate was able to control fludioxonilresistant isolates, ‘Eureka’ lemons were wounded with a stainless steel rod (1 × 2 mm) and inoculated with 10 μl of conidial suspensions (6 × 10 conidia/ml) of a representative sensitive or a resistant isolate. After 4 h, inoculated fruit were dipped for 30 s in either a formulated product of fludioxonil, Graduate (50% active ingredient; Syngenta Crop Protection, Research Triangle Park, Raleigh, NC) at 1,198 mg/liter, or water as a control, and then stored at 20°C in air for 7 days. There were four 20-fruit replicates for each treatment and the experiment was performed twice. All inoculated water-dipped fruit were decayed, while fludioxonil completely controlled green mold on fruit inoculated with the fludioxonil-sensitive isolate. However, 100% fruit inoculated with the fludioxonil-resistant isolate and treated with fludioxonil were decayed. Fludioxonil-insensitive isolates have been reported to occur in natural populations of P. digitatum before its commercial use, but at very low frequency (1.4 to 2.5 × 10) (Kanetis et al. 2006). This is the first report of fludioxonil resistance in P. digitatum collected from commercial citrus packinghouses after the introduction of the fungicide on the market. These fludioxonil-resistant isolates were obtained from packinghouses where the fungicide had been used during citrus packing for 2 consecutive years, approximately for a 3-month period in each year, indicating that fludioxonil-resistant individuals emerged quickly in P. digitatum populations. Our results show that fludioxonil failed to control green mold incited by the fludioxonil-resistant isolate, suggesting that appropriate fungicide resistance management practices need to be implemented to ensure adequate control.

Postharvest efficacy of resistance inducers for the control of green mold on important Sicilian citrus varieties

The disease reducing activity of acibenzolar-S-methyl (ASM), chitosan and β-aminobutyric acid (BABA) against postharvest green mold (GM) of citrus fruit, Penicillium digitatum, was investigated by tests performed in in vitro and in vivo conditions. The inhibition of the mycelial growth of P. digitatum by ASM, chitosan and BABA at different concentrations was evaluated on potato dextrose agar and on orange peel extract agar. The pathogen was totally inhibited (100% growth inhibition) by chitosan at concentrations ≥ 0.1% and by BABA at 1000 mmol and only poorly inhibited by ASM at 0.5% (47% growth inhibition). Inhibitory effects of chitosan and BABA were confirmed by P. digitatum conidial germination tests. The performances of these chemical compounds were evaluated in vivo on artificially inoculated orange, lemon and grapefruit cultivars. On the whole, chitosan significantly reduced GM decay on ‘Valencia’ and ‘Washington Navel’ orange, ‘Femminello’ lemon and grapefruit cv. Marsh Seedless at the concentration of 0.5% (6–20% disease incidence), and BABA provided the best results on ‘Tarocco Scire’and ‘Valencia’ orange and grapefruit cv. Marsh Seedless at the concentration of 1000 mmol (12–16% disease incidence). ASM did not show any efficacy. This study demonstrated that the effects of chitosan and BABA on GM in citrus fruits may be associated with their direct fungitoxic properties against the pathogen. Treatments with chitosan and BABA could be recommended for inclusion in postharvest decay management programs for citrus packinghouses and their use may be an effective method to improve the integrated pest management strategy.

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±0.03mgL−1 while the mean EC50 of those collected from packing houses was 0.13±0.05mgL−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 3.40mgL−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 500–600mgL−1 by immersion for 60s at room temperature to inoculated oranges and mandarins. In contrast, the resistant isolate was not controlled by PYR applied at 1000mgL−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.

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 6L per 100m3 of room volume dispensed over a 3h period. Rates applied were expressed as active ingredient per m3 of room volume. All were compared to formaldehyde applied at 1.98gm−3 of room volume. After 24h, 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 (0.1gm−3); (2) hydrogen peroxide (4.4gm−3); (3) Citrisol (1.0gm−3), a proprietary mineral oxychloride oxidizer; (4) acetic acid (5.3gm−3); and (5) peracetic acid (2.4gm−3). The toxicity of effective sanitizers was determined by exposure of P. digitatum conidia for 10min 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.

Evaluation of postharvest treatments with chemical resistance inducers to control green and blue molds on orange fruit

Preventive and curative activities of postharvest treatments with selected chemical resistance inducers to control postharvest green (GM) and blue (BM) molds on oranges (cvs. ‘Valencia’ or ‘Lanelate’) artificially inoculated with Penicillium digitatum and Penicillium italicum, respectively, were evaluated. In vivo primary screenings to select the most effective chemicals and concentrations were performed with benzothiadiazole (BTH), β-aminobutyric acid (BABA), 2,6-dichloroisonicotinic acid (INA), sodium silicate (SSi), salicylic acid (SA), acetylsalicylic acid (ASA) and harpin. INA at 0.03mM, SA at 0.25mM, BABA at 0.3mM and BTH at 0.9mM were selected and tested afterwards as dips at 20°C for 60 or 150s with oranges artificially inoculated before or after the treatment and incubated for 7d at 20°C. Although it was an effective treatment, SSi at 1000mM was discarded because of potential phytotoxicity to the fruit rind. Preventive or curative postharvest dips at room temperature had no effect or only reduced the development of GM and BM very slightly. Therefore, these treatments cannot be recommended for inclusion in postharvest decay management programs for citrus packinghouses.

Antifungal activity of extracts of extremophile plants from the Argentine Puna to control citrus postharvest pathogens and green mold

A main constraint on the citrus industry is the management of postharvest diseases, mostly of fungal origin. The fungicides used to control these diseases: (1) leave persistent residues that raise some dietary concerns; (2) cannot be used on fruit produced under ‘organic’ rules; and, (3) have experienced resistance to them become widespread within citrus packinghouses. Consequently, the development of management strategies to supplement or replace the use of synthetic fungicides by the utilization of microorganisms or natural bioactive products is desirable. The aim of this work was to screen extremophile plants from the Argentine Puna in order to select species with antifungal activity against Penicillium digitatum and Geotrichum citri-aurantii, two very important fungal species causing citrus postharvest disease. Plant aqueous extracts obtained from Chuquiraga atacamensis, Parastrephia phyliciformis and Parastrephia lepidophylla, were able to inhibit in vitro P. digitatum growth, while P. lepidophylla extract was active against G. citri-aurantii cultures. P. lepidophylla aqueous formulations showed MIC100 values of 300mg/L against both P. digitatum and G. citri-aurantii strains. MFC values of P. lepidophylla extracts were similar to MIC100 values. In vivo tests showed that the P. lepidophylla aqueous formulations (600mg/L) exert curative and protective effects on the fruit against infection by a P. digitatum wild strain.

Imazalil concentration for in vitro monitoring of imazalil resistant isolates of Penicillium digitatum in citrus packinghouses

The extensive use of imazalil (IMZ) in Uruguayan citrus packinghouses to control Penicillium spp. favored the selection and proliferation of resistant isolates. With the aim of detecting Penicillium digitatum biotypes that are not controlled by commercial doses of IMZ, the IMZ concentration within amended potato dextrose agar (PDA) plates was adjusted to 1.0 mg L −1 IMZ. This concentration allowed the detection of resistant isolates that were not controlled by commercial applications of 3.0 g L −1 IMZ. These isolates were able to grow in fruit with IMZ residues of 0.92 and 3.08 mg kg −1. Therefore, environmental monitoring of facilities where commercial dip applications containing 3.0 g L −1 IMZ are employed, should be done with 1.0 mg L −1 of IMZ in Petri dishes. We can conclude that use of IMZ in postharvest applications of 3.0 g L −1 remains effective to control wild Uruguayan P. digitatum isolates but not for resistant ones. A survey of 26 P. digitatum isolates collected in Uruguay indicated IMZ resistant isolates occurred in packinghouses and not in citrus groves.

Control of citrus postharvest decay by ammonia gas fumigation and its influence on the efficacy of the fungicide imazalil

Postharvest green mold and blue mold, caused by Penicillium digitatum and Penicillium italicum, respectively, were effectively controlled by fumigation of lemons and oranges for 6 h at 22 °C with two applied dosages of 3000 μL L −1 of ammonia that was injected initially and again 2 h later. This treatment did not injure oranges, however, it caused the tissue within previously injured areas on the rind of lemons to become darker in color. Fumigation of lemons with 6000 μL L −1 of ammonia slightly accelerated the natural transition of rind color from green to yellow. The germination of conidia of P. italicum was more sensitive to ammonia than those of P. digitatum, although many survived fumigation. About 30% of the conidia of P. digitatum and 10% of those of P. italicum could germinate after a 6 h fumigation where two injections of 6000 μL L −1 of ammonia were applied, one initially and a second 2 h later. Ammonia fumigation controlled an isolate of P. digitatum with a high level of resistance to imazalil (IMZ). The influence of ammonia fumigation on the effectiveness of this common postharvest fungicide was examined. When fruit were first immersed in 10 or 30 mg L −1 IMZ (about 10% of typical commercial rates) before ammonia fumigation, a single fumigation with 1500 μL L −1 of ammonia was adequate to control both diseases and the increase in effectiveness was usually additive and sometimes synergistic. This effect was probably due in part to the influence of pH on IMZ activity, because the neutral form of IMZ increases with increasing pH and it has markedly higher antifungal activity than the ionized molecule. Fumigation with 1500, 3000, or 6000 μL of ammonia per liter increased the pH (±SD) of albedo tissue of pre-existing wounds on oranges and lemons from 5.9 (±0.2) before fumigation by 0.6 (±0.3), 0.9 (±0.4), or 1.3 (±0.3) units, respectively. IMZ can be applied immediately after harvest by drenching fruit within harvest bins with aqueous IMZ solutions. Subsequent ammonia fumigation on their arrival to packinghouses may be a feasible practice, since it could employ the existent ethylene degreening chambers present at all packinghouses, if these were modified to be gas tight. Ammonia could replace synthetic fungicides or augment IMZ performance in citrus postharvest decay management. Its capacity to control IMZ resistant isolates of P. digitatum, common in citrus packinghouses, is particularly valuable.

Determination of Natural Resistance Frequencies in Penicillium digitatum Using a New Air-Sampling Method and Characterization of Fludioxonil- and Pyrimethanil-Resistant Isolates

ABSTRACT Fungicide resistance was identified in natural populations of Penicillium digitatum, the causal agent of green mold of citrus, to two of three new postharvest fungicides before their commercial use. Using a new air-sampling method where large populations of the pathogen in citrus packinghouses were exposed to agar plates with a continuous, wide-range fungicide concentration gradient, isolates with reduced sensitivity to fludioxonil or pyrimethanil were obtained. Resistance frequencies to fludioxonil and pyrimethanil were calculated as 9.5 x 10(-7) to 1.5 x 10(-5) and 7.3 x 10(-6) to 6.2 x 10(-5), respectively. No isolates resistant to azoxystrobin were detected. Isolates with reduced sensitivity to fludioxonil or pyrimethanil were also obtained in laboratory selection studies, where high concentrations of conidial mixtures of isolates sensitive to the three fungicides were plated onto agar amended with each fungicide at 10 microg/ml. Isolates obtained from fludioxonil selection plates in laboratory and packinghouse experiments were placed into two categories based on mycelial growth: moderately resistant isolates had 50% effective concentration (EC(50)) values of 0.1 to 0.82 microg/ml and highly resistant isolates had EC(50) values > 1.5 microg/ml. Isolates resistant to pyrimethanil all had EC(50) values >8 microg/ml. Representative isolates of the two categories with reduced sensitivity to fludioxonil varied widely in their virulence and sporulation capacity as measured by the incidence of decay and degree of sporulation on inoculated fruit, respectively, whereas pyrimethanil-resistant isolates were mostly similar to the wild-type isolate. Fungicide sensitivity characteristics for isolates from fludioxonil and pyrimethanil selection plates remained stable after passages on nonamended agar, and disease could not be controlled after treatment with the respective fungicides. Types of fungicide resistance were visualized on thiabendazole- (TBZ) and imazalil-amended selection plates that were exposed in packinghouses where resistance to these fungicides was known to occur. The qualitative, single-site resistance to the benzimidazole TBZ was visualized by two distinct subpopulations in regard to fungicide sensitivity, whereas the quantitative, multi-site resistance to the demethylation inhibitor imazalil was apparent as a continuous density gradient of colonies along the fungicide concentration gradient. Types of resistance could not be assigned to fludioxonil or pyrimethanil because a limited number of resistant colonies was obtained on each plate. Thus, with this new method, we were able to estimate fungicide resistance frequencies as well as characterize and visualize types of resistance within populations of a fungal species. This information will be used to design resistance management strategies for previous and newly registered postharvest fungicides of citrus.

Characterisation of the fungal population in citrus packing houses

The aim of this work was the characterisation of the environmental and superficial mycoflora of equipment and facilities of two citrus packing houses in Sao Paulo State, Brazil, in 2004 and 2005. One of the packing houses packed fruit for the export market (municipality of Matao), the other for the domestic market (municipality of Engenheiro Coelho). The study also identified the presence of isolates of Penicillium spp. resistant to thiabendazole and imazalil fungicides in packing houses. The environmental mycoflora was sampled according to the gravimetric method, using Petri dishes containing potato dextrose agar medium opened for 2 min. The superficial mycoflora on equipment and facilities was sampled with Rodac plates. The mycoflora in the environment and on surfaces of the packing houses in Matao were 12.3 and 52.3 cfu/plate, respectively, while these populations for the Engenheiro Coelho packing house were 46.3 and 68.2 cfu/plate, respectively. Cladosporium and Penicillium were the most prevalent genera of fungi. The contamination levels of clean zones in the packing houses (washing of fruits, packing table, boxes and containers) was not substantially lower than the contamination in dirty zones (reception of fruits and first selection). The percentage of P. digitatum isolates in Matao that was resistant to thiabendazole and imazalil was 25.9 and 1.5 in the environment and 30.1 and 16.0 on packing house surfaces, respectively. In Engenheiro Coelho, percentage of resistance to these fungicides was 51.9 and 0.1 in the environment and 39.2 and 0.9 on packing house surfaces, respectively.

Preventive and curative activity of combined treatments of sodium carbonates and Pantoea agglomerans CPA-2 to control postharvest green mold of citrus fruit

Preventive and curative activity of 2 min dips in 3% sodium carbonate (SC) or sodium bicarbonate (SBC) aqueous solutions heated to 40 °C, alone or followed by the application of 2 × 10 8 CFU/mL of the biocontrol agent Pantoea agglomerans CPA-2 (BA), were simultaneously evaluated for the control of postharvest green mold, caused by Penicillium digitatum, in artificially inoculated Lanelate and Valencia oranges. Fresh cells of BA proliferated inside rind wounds and their survival was not adversely affected by the presence of residues of SC or SBC. Green mold incidence after 7 d of incubation at 20 °C in rind wounds treated after fungal inoculation (curative activity) was 15%, 40%, or 15% in oranges treated with SC, BA, or SC + BA and 5%, 45% or 0% in oranges treated with SBC, BA, or SBC + BA, respectively, while it was about 90% in untreated control fruit. Green mold incidence in rind wounds treated before inoculation or reinoculation with the pathogen (preventive activity in pre-existing wounds) was 10% and 2%, or 15% and 8%, respectively, in oranges treated with SC and SC + BA, and 3% and 5%, or 20% and 5%, respectively, in oranges treated with SBC and SBC + BA. Green mold incidence in wounds inoculated after treatment (preventive activity in new wounds) was 55% and 25%, and 60% and 40% in oranges treated with SC and SC + BA, or SBC and SBC + BA, respectively. Additionally, the duration of the protective effect of SBC, BA, and SBC + BA was assessed in Eureka lemons and Valencia oranges. In both species, all three treatments effectively protected pre-existing rind wounds during 7 d of storage at 10 °C. After 0, 1, and 2 d, but not after 4 or 7 d, the protective effect of SBC was significantly inferior to that of BA and SBC + BA. The integration of treatments is a promising approach to replace the use of conventional fungicides to control green mold in citrus packinghouses.

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.

Hyperspectral detection of citrus damage with Mahalanobis kernel classifier

Presented is a full computer vision system for the identification of post-harvest damage in citrus packing houses. The method is based on the combined use of hyperspectral images and the Mahalanobis kernel classifier. More accurate and reliable results compared to other methods are obtained in several scenarios and acquired images.

Mutation at β-Tubulin Codon 200 Indicated Thiabendazole Resistance in Penicillium digitatum Collected from California Citrus Packinghouses

Thiabendazole (TBZ) is commonly applied to harvested citrus fruit in packinghouses to control citrus green mold, caused by Penicillium digitatum. Although TBZ is not used before harvest, another benzimidazole, thiophanate methyl, is commonly used in Florida and may be introduced soon in California to control postharvest decay of citrus fruit. Isolates from infected lemons and oranges were collected from many geographically diverse locations in California. Thirty-five isolates collected from commercial groves and residential trees were sensitive to TBZ, while 19 of 74 isolates collected from 10 packinghouses were resistant to TBZ. Random amplified polymorphic DNA analysis indicated that the isolates were genetically distinct and differed from each other. Nineteen TBZ-resistant isolates and a known TBZ-resistant isolate displayed a point mutation in the β-tubulin gene sequence corresponding to amino acid codon position 200. Thymine was replaced by adenine (TTC → TAC), which changed the phenylalanine (F) to tyrosine (Y). In contrast, for 49 TBZ-sensitive isolates that were sequenced, no mutations at this or any other codon positions were found. All of the isolates of P. digitatum resistant to TBZ collected from a geographically diverse sample of California packinghouses appeared to have the same point mutation conferring thiabendazole resistance.