Evaluation of Prophage Gene Revealed Population Variation of ‘Candidatus Liberibacter Asiaticus’: Bacterial Pathogen of Citrus Huanglongbing (HLB) in Northern Thailand

‘Candidatus Liberibacter asiaticus’ is a non-culturable bacterial pathogen, the causal agent of Huanglongbing (HLB, yellow shoot disease, also known as citrus greening disease), a highly destructive disease of citrus (Rutaceae). The pathogen is transmitted by the Asian citrus psyllid: Diaphorina citri Kuwayama. Recent studies, have shown that the HLB pathogen has two prophages, SC1 that has a lytic cycle and SC2 associated with bacterial virulence. This study aimed to search for SC1 and SC2 prophages of HLB in mandarin orange, sweet orange, bitter orange, kumquat, key lime, citron, caviar lime, kaffir lime, pomelo and orange jasmine from five provinces in Northern Thailand. A total of 216 samples collected from Northern Thailand during 2019 and 2020 were studied. The results revealed that 62.04% (134/216) citrus samples were infected with the ‘Ca. L. asiaticus’ the bacterial pathogen associated with citrus HLB. The prophage particles are important genetic elements of bacterial genomes that are involved in lateral gene transfer, pathogenicity, environmental adaptation and interstrain genetic variability. Prophage particles were evaluated in the terminase gene of SC1 and SC2-type prophages. The results of the analysis of the prophage terminase genes of SC1 and SC2 revealed four groups of samples in Northern Thailand.The first group was the population of ‘Ca. L. asiaticus’ which was non-prophage; the frequency was 7.46% (10/134) from the total infected citrus samples. The second and third groups contained one prophage sequence with 1.49% (2/134) containing SC1-type prophage and 64.93% (87/134) containing SC2-type prophage. The fourth group contained both prophage sequence with 26.12% (35/134) containing SC1 and SC2-type prophages.Samples of leaves showing various HLB symptoms were collected from infected citrus trees which correlated with the prophages. In the first group (non-prophage) the symptoms were much less severe than other groups which had prophages. On the other hand, the third group containing the SC2-type prophage and fourth group containing SC1 and SC2-type prophages showed the most severe symptoms. The evaluation of the prophage effect on HLB-induced leaf symptoms showed that isolates with the SC2-type prophage particles produced the most severe symptoms.

Unusual Stylar-End Breakdown and Sour Rot on Key Lime (Citrus aurantiifolia) in Pre-Harvest Condition in Italy

Key lime (Citrus aurantiifolia) is an emerging crop in Italy, especially in the Southern regions, where the environmental conditions are suitable for its cultivation. A field survey in Sicily in a commercial orchard of Key lime revealed the widespread presence of water-soaked spots and sunken/dry lesions at the stylar-end, mainly in pre-harvest condition. Water-soaked spots were attributed to Geotrichum citri-aurantii, an agent of sour rot on Citrus spp., whereas the sunken/dry lesion was attributed to the physiological disorder known as stylar-end breakdown. Sour rot and stylar-end breakdown are usually considered post-harvest diseases and rarely found in the field on fruit still attached to the tree. Although Geotrichum citri-aurantii is not responsible for the stylar-end breakdown, its association with this alteration reveals the importance of the environmental conditions and the agronomic practices in diseases/disorders development. In addition, to our knowledge, this is the first report of Geotrichum citri-aurantii on Key lime in Europe.

Fungal Endophytes of Tahiti Lime (Citrus citrus × latifolia) and Their Potential for Control of Colletotrichum acutatum J. H. Simmonds Causing Anthracnose

Colletotrichum acutatum is one of the causal agents of anthracnose in several crops, and of post-flowering fruit drop (PFD) in citrus and key lime anthracnose (KLA). The pathogen normally attacks flowers, causing lesions only in open flowers. Under very favorable conditions, however, it can also affect flower buds and small fruits, causing complete rotting of the fruit and a premature fall, resulting in major economic crop losses. We isolated endophytic fungi from Tahiti lime to evaluate its diversity, verify its antagonistic capacity against the phytopathogen Colletotrichum acutatum C-100 in dual tests, and evaluate the ability of various endophytic agents to control flowers with induced anthracnose. 138 fungal isolates were obtained from 486 fragments of branches, leaves, and fruit; from which 15 species were identified morphologically. A higher isolation frequency was found in branches and leaves, with a normal level of diversity compared to other citrus species. Of the 15 morphospecies, 5 were trialed against C. acutatum in antagonism tests, resulting in a finding of positive inhibition. 2 endophytic fungi from the antagonism tests demonstrated high inhibition of the phytopathogen, and were thus used in in vivo tests with Tahiti lime flowers, applied in a spore solution. Spore solutions of two molecularly identified species, Xylaria adscendens, and Trichoderma atroviride, reduced the lesions caused by the phytopathogen in these in vivo tests. The finding that these endophytes react antagonistically against C. acutatum may make them good candidates for further biological control research in an agroindustry that requires environmental sustainability.

Infrared Radiation Favorably Influences the Quality Characteristics of Key Lime Juice

The effect of infrared radiation (IR) on the physicochemical characteristics, pectin methylesterase activity (PME), hydroxymethylfurfural (HMF) content, microbiological activity, color, and sensory aspects on black lime juice was studied. IR was compared to conventional thermal heating (CTH) in batch infrared extraction pasteurizer, designed to allow both infrared and conventional heating. IR resulted in a reduction in pH and Brix values and a mild increase in titratable acidity, as compared to CTH and control. After 60 days at 5 °C, the ascorbic acid percentage was decreased by 24.90%, 29.75%, and 58.31% in the control, IR and CTH, respectively. The total amount of phenols in juice treated with IR was higher as compared to CTH and control, while there was a significant decrease in the antioxidant activity. The statistical analysis reflected significantly low (p < 0.05) activity of PME for IR samples as compared to CTH and control. The amount of Hydroxymethylfurfural (HMF) in all juice samples steadily increased during the storage at 5 °C in 60 days. The microbial content of control was 3.85 log cfu/mL after 60 days at 5 °C, while it was 2.1 log cfu/mL for IR which reflected a significant difference between the IR, CTH, and control samples. Additionally, color and sensory analysis of IR treated sample when compared to control, reflected similar attributes. Overall, IR was found to be an excellent substitute for the preservation of black lime juice as a rapid pasteurization technique with less heat exposure; wherein the nutrition and health benefits of the juice could be maintained for a minimum period of 60 days.

Bioactive Substances, Heavy Metals, and Antioxidant Activity in Whole Fruit, Peel, and Pulp of Citrus Fruits

The use of whole citrus fruits in the food industry means that the valuable peel is used, but this may raise palatability or health concerns among consumers. The content of sugars, dietary fibre, redox compounds, lead, and cadmium was compared in citrus fruits (orange; pomelo; mandarin; lemon; key lime; and red, yellow, and green grapefruit). The pulp of all fruits contained significantly less fibre, tannins, and phenolic compounds than the peel. Whole citrus fruits had significantly lower content of sugars and higher content of dietary fibre and phenolic compounds, including ferulic acid, than their pulps. Whole grapefruits had higher concentrations of ascorbic acid. Whole lemons, limes, and mandarins had higher antioxidant potential than their pulp, due to their higher content of ascorbic acid, tannins, and phenolic compounds. Lead and cadmium content in whole fruits, while higher than in the pulps, was well below the acceptable daily intake.

Swelling, Tensile and Thermal Behaviors of Citric Acid Crosslinked Tapioca Starch/Cellulose Biocomposite Films

The biocomposite films were prepared using a solution casting method and allowed to dry in the oven of 50°C. The cellulose used in this research was extracted from the rice straw. The biocomposite films firstly were prepared without crosslinker with various cellulose content; 0.2g, 0.4g, 0.6g, 0.8g, and 1.0g wt%. Based on the tensile strength result, 0.6g of cellulose is an optimum amount to prepare the biocomposite films with various amounts of crosslinker; 1g, 2g, 3g, 4g, and 5g. The tapioca starch/cellulose biocomposite films crosslinked with lemon juices have higher tensile strength (16MPa) and lower in swelling percentage (3.32%) compared to the biocomposite films crosslinked with key lime juice (5.44%). The thermal behavior was studied based on the Differential Scanning Calorimetry test shows the biocomposite film that highly crosslinked needs higher energy during their phase change. The energy liberated in the tapioca starch/cellulose biocomposite film with key lime juice as crosslinker is 201.6 J/g while that of biocomposite film with lemon juice as crosslinker is 383.0 J/g.