Studies On The Mechanisms Of DNP And ATP Regulating The Disease Development Of Harvested Longan Fruit Infected By Lasiodiplodia Theobromae

Longan(Dimocarpus longan Lour.)is a featured fruit in the south of China,and also planted globally in tropical and subtropical countries or areas.It has been favored by the consumers since ancient times for its high nutrient values,hence promising high commercial potential.The maturation and harvest periods of longan fruit are in August and September with high temperature and humidity,making the fruit vulnerable to pathogen infection and consequent fruit rot characterized by pericarp browning and aril breakdown.This gives severe restriction to the remote transportation and marketing,as well as long-term storage of harvested longan fruit.Thus,in order to prevent and control the postharvest disease of longan fruit,there is a need to investigate the interaction between pathogen invasion and the fruit.Lasiodiplodia theobromae(Pat.)Griff.&Maubl.(L.theobromae)was found to be a key pathogen fungus leading to longan fruit’s postharvest disease.Previous studies demonstrated that pathogen infection could result in the decrease of adenosine triphosphate(ATP)content and energy charge in the pericarp of harvested longans;additionally,energy status was proved to be capable of regulating the pericarp browning of harvested longans.Therefore,it could be inferred that pathogen infection was believed to be a critical factor causing the change in energy status which could further affect the pericarp browning and disease development in longan fruit during storage.However,literatures to date rarely involve the effects of energy level on the postharvest disease development of longans or the underlying physiological mechanism.Therefore,upon previous work,this effort investigated the effects of 2,4-dinitrophenol(DNP)and ATP on the disease development of harvested longan fruit caused by L.theobromae,regarding the metabolisms of energy,respiration,reactive oxygen,antifungal substance and cell wall material with ’Fuyan’ longan fruit as matrial.The objectives of this paper were to put forward a possible mechanism for harvested longan fruit disease development,and to provide the scientific theoretical foundation and guidance to control longan fruit’s postharvest disease.The main results are listed below:1.DNP treatment induced an accelerated increase in the indices of disease,browning and aril breakdown in L.theobromae-inoculated longans,indicating that DNP could promote the disease development of infected fruit;ATP treatment,on the contrary,inhibited the rise of disease,browning and aril breakdown indices in L.theobromae-inoculated fruits,showing the delaying effect on the disease development.2.For the pericarp of L.theobromae-inoculated longans,DNP treatment stimulated the decline of ATP and ADP contents,as well as energy charge,inhibited the activities of Ca2+-ATPase,Mg2+-ATPase and H+-ATPase across the membrane of protoplasm,vacuole and mitochondria,leading to inefficient ATP synthesis,and unbalanced cellular Ca2+and H+ concentrations,and eventually resulting in altered osmotic pressure of membrane and membrane structure damage.ATP treatment could delay the decrease in the level of ATP and energy charge,kept the activities of ATPase on protoplasm,vacuole and mitochondria membranes to maintain the ion balance,protection the membrane structure and function.3.For the L.theobromae-inoculated fruits,DNP treatment elevated the increase in fruit respiration rate and the activities of pericarp respiratory terminal oxidase including cytochrome C oxidase(COX),ascorbic acid oxidase(AAO)and polyphenol oxidase(PPO),as well as activities of phosphohexose isomerase(PGI)and succinodehydrogenase(SDH),but strengthened the inhibition on the total activity of 6-phosphaogluconate dehydrogenase(6-PGDH)and glucose-6-phosphate dehydrogenase(G-6-PDH),as well as the activity of nicotinamide adenine dinucleotide kinase(NADK),maintained the level of oxidized form of nicotinamide adenine dinucleotide(NAD)and its reduced form(NADH),thus restrained the content of oxidized form of nicotinamide adenine dinucleotide phosphate(NADP)and its reduced form(NADPH),resulting in impaired pentose phosphate pathway which is highly related to pathogen-infection-response.On the other hand,ATP treatment notably delayed the increase in the fruit respiration rate and in the activities of pericarp terminal oxidase of L.theobromae-inoculated fruits,inhibited the activities of PGI and SDH,but maintained G-6-PDH and 6-PGDH activity,accelerated the rise of NADK activity to enhance the decline of NAD and NADH contents,and the increased in NADP and NADPH contents,reinforced pentose phosphate pathway.4.For the pericarp of L.theobromae-inoculated fruits treated with DNP,the O2-· production rate and malondialdehyde(MDA)content were largely promoted;the activities of reactive oxygen scavenging enzymes like superoxide dismutase(SOD),catalase(CAT)and ascorbate peroxidase(APX)were suppressed;the reduce of reactive oxygen scavenging substances such as ascorbic acid(AsA)and glutathione(GSH)were further stimulated,and the 1,1-diphenyl-2-picrylhydrazyl(DPPH)scavenging ability and total reduction ability were further decreased,resulting in severe over-accumulation of reactive oxygen species(ROS)and higher level of membrane peroxide.On the contrary,there were inhibited increase of O2-· roduction rate and MDA content,elevated activities of reactive oxygen scavenging enzymes and substances,and relatively higher DPPH scavenging ability and reduction ability in pericarp of L.theobromae-inoculated fruits with ATP treatment,maintained the balance between ROS production and scavenging.5.DNP treatment stimulated the increase in the activities of fatty acid degrading enzymes including phospholipase D(PLD),lipase and lipoxygenase(LOX)in the pericarp of L.theobromae-inoculated fruits,accelerated the degradation of membrane phospholipids like phosphatidylcholine(PC)and phosphatidylinositol(PI),as well as the degradation of unsaturated fatty acids like linoleic acid(C18:2),linolenic acid(C18:3)and gondoic acid(C20:1),promoted the increase of phosphatidic acid(PA),as well as the relative contents of saturated fatty acids like palmitic aicd(C16:0)and stearic acid(C18:0),consequently aggravated the decreasing ratio of unfatty acid to fatty acid(U/S)with an enhanced rise of membrane permeability,leading to the membrane structural damage;ATP treatment could inhibit the activities of fatty acids degrading enzymes,alleviate the degradation of phospholipids and unfatty acid,delay the increase of fatty acid,maintain higher index of unfatty acid(IUFA),and delayed the change of fatty acid components to protect the membrane integrity.6.DNP treatment could inhibit the rise of total phenolics content,and strengthen the increase in the activities of phenylalanine ammonia-lyase(PAL)and peroxidase(POD)in pericarp of L.theobromae-inoculated fruits during initial storage period;while during later storage,DNP treatment aggravated the decrease of total phenolics content,inhibited the PAL activity,but further enhanced the increase of POD activity to accelerate pericarp browning.Additionally,DNP treatment inhibited the increase of lignin,promoted the reduction of flavonoids,and suppressed the activities of anti-pathogen enzymes like chitinase(CHI)and β-1,3-glucanase(GLU)),resulting in decreased disease resistance of L.theobromae-inoculated longans.ATP treatment inhibited the activites of PAL and POD,and kept low content of lignin in pericarp of L.theobromae-inoculated fruits during the initial storage;while during the later storage,it increased the activities of PAL,facilitated the synthesis of lignin,and elevated the POD activity.Moreover,ATP treatment also delayed the decline of flavonoids and total phenolics contents,increased the activities of CHI and GLU,leading to maintained disease resistance of L.theobromae-inoculated fruits.7.DNP treatment enhanced the rise of cell wall degrading enzymes activities including polyethylene(PE),polygalacturonase(PG),cellulase(Cx)and β-galactosidase in pericarp of L.theobromae-inoculated fruits,aggravated the degradation of cell wall materials and the transformation of covalent-bound pectin to ionic-bound pectin and water-soluble pectin,accelerated the decrease of cellulose and hemicellulose contents,causing severe cell wall structure damage;ATP treatment delayed the increase of activities of PE,PG,Cx and β-galactosidase in pericarp of L.theobromae-inoculated fruits,alleviated the degradation of cell wall materials like pectin,cellulose and hemicelluloses,so as to maintain the cell wall structure.