| China is the largest citrus-producing country around world while the loss from post-harvest decay is considerable. Conventional chemical fungicides, which once was a worldwide use, has been increasingly curtailed by the development of pathogen resistance to many key fungicides caused by its excessive use and misuse, less eco-friendly, and public perception that they are harmful to human health and environment. Thus, alternative to conventional chemical fungicides for the control of citrus post-harvest diseases are urgently needed. Biological control agents (BCAs) herein give a promising choice.Kloechera apiculata is usually mentioned as a brewing yeast, yet, according to our study, it also possesses the trait of citrus post-harvest diseases control. K. apiculata strain 34-9, isolated from natural citrus garden, has been demonstrated the quality of effective protection citrus fruits against wound pathogens. We mainly did our research in three aspects:evaluation of yeast apoptosis due to induction of acetic acid, morphological features and apoptotic phenotype in K. apiculata 34-9 during cells preparation and cold storage by means of scanning electron microscopy, transmission electron microscopy, pulsed field gel electrophoresis, TUNEL reaction and other fluorescent staining, like DHE, Annexin V-FITC and PI.1. Served as an anti-microbial preservative, acetic acid can remarkably inhibit the proliferation of antagonist 34-9 at 20 mM, while the culture density hardly changes under 40 mM acetic acid after 3 hs'incubation. By the examination of cell death markers that are typical of apoptosis, we confirmed that acetic acid could trigger an apoptotic phenotype in Kloeckera apiculata 34-9. The following changes were observed after treatment with 40 or 60 mM acetic acid:chromosomal DNA fragmentation as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining; exposure of phosphatidylserine on the surface of cytoplasmic membrane; DNA breakdown into fragments mainly consisted of single-and/or double-strand DNA breaks, which were consistent with the higher order chromatin degradation preceding DNA laddering in apoptotic mammalian cells; accumulation of ROS; disruption of the mitochondrion, namely disappearance of cristae, formation of swelling bodies and dissolution of homogenization of nucleus and nuclear membrane.2. It was shown that antagonist 34-9 catabolyzed glucose during early fermentative process and produced plenty of ethanol, leading to the conclusion that yeast 34-9 possessed crabtree effect, namely occurrence of alcoholic fermentation under aerobic conditions rather than cell growth. This helps us understand why even though there are abundant nutrients available, the biomass rarely seldom be greater than 108/mL during cells preparation. Otherwise, glucose represses the assimilation of ethanol and acetate in yeast, while large accumulation of ethanol and acetate has. a negative impact on yeast fermentative performance. In the line of this statement, instead of blindly increasing concentration of glucose to produce cells, most of our attention should be given to its role in the carbon flux regulation. The percentage of apoptosis is low at 24 or 48 h of batch culture. It is likely that the concentration of metabolites is not high enough to trigger the apoptosis in cells. The acid stress-adaptation can protect cells from acetic acid-induced cell death and improve cell viability.3. Citrus storage environment is extremely unfavorable for the survival of yeast 34-9 which begins to die mainly in the mode of non-apoptosis in the early stage of storage. Low viability may be related to the absence of pathogens, because as the natural enemy to pathogens, antagonistic yeasts may survive on them, which serves as one factor of effective antagonism. |
PDF Full Text Request