Research progress in citrus melanose

Abstract: Citrus (Citrus spp.) is the world’s largest category of fruit, including many species, such as C. reticulata, C. sinensis, C. grandis, C. limon and C. paradisi, etc.. They are deeply popular with consumers, because of their rich nutrition (vitamins, polysaccharides, organic acids, proteins, dietary fiber and antioxidants) and delicious flavor. The global citrus production for 2019 was estimated at almost 144 million metric tons. In China, the citrus production for 2021 was 55.96 million metric tons, and its annual value of production was more than 200 billion yuan. Therefore, it is the vital major industry of agriculture in China. However, in recent years, the citrus melanose has seriously occurred in major citrus production regions all over the world, including China, India, Brazil, Spain and Mexico, etc.. In China, the citrus melanose was widely distributed in Guangxi, Hunan, Hubei, Zhejiang, Jiangxi, Fujian, Yunnan provinces and other major citrus production areas, and the disease incidences in some orchards were up to 100%. The typical disease symptoms including melanose, gummosis and stem-end rot form on leaves, shoots or fruits. The diseased fruits usually show many black to reddish-brown raised spots on the fruit surface or stem-end rot, which seriously affects the appearance and economic value of fresh fruit, causing major economic losses. Diaporthe citri (anamorph: Phomopsis citri) is the dominant species of citrus melanose pathogen all over the world, which could infect all citrus cultivars. At present, the genome information of D. citri has been sequenced and annotated, which provides an important reference for studying its infection mechanism and population genetic evolution, etc.. The genomes of D. citri (MAT1-1 and MAT1-2 strains) contains 15 977-16 622 genes, including 1231-1287 putative pathogenicity genes, 1837- 1885 secretion proteins, and many carbohydrate- active enzymes (CA-Zymes), which may be associated with the pathogenicity of D. citri. The populations of D. citris are abundant in genetic diversity, due to its frequent sexual reproduction in nature. And the genetic differentiation of D. citris is closely related to geographic separation, whereas it has weak correlation with its host. The species-specific primers have been designed for the PCR method to distinguish D. citri from related Diaporthe species, based on the sequences of rDNA internal transcribed spacer, translation elongation factor 1- alpha, beta-tubulin, histone H3, and calmodulin gene, which contribute to monitoring and forecasting the citrus melanose in the field. It has been reported that the successful infection host by D. citris is related to its pectinase secretion, and the infection of leaves can cause an increase in the population of antagonistic microorganisms in the citrus leaves. The RNA-Seq analysis results performed by Li et al. profiled the defense response pattern of citrus leaves against D. citri infection, including high induction expression of plant cell wall biogenesis-related genes at 3 days post infection (dpi), and high upregulation expression of the CYP83B1 genes, pectin methylesterase gene, and phytoalexin coumarin synthesis-related genes at 14 dpi. After the infected shoots became withered, a large number of alpha conidia (non-septate), beta conidia (long, slender) and a small number of ascospores (ellipsoid to cylindrical, septate) were produced on dead wood, using as the source of infection. Conidia were carried by raindrops and dispersed to nearby citrus, which contributed obviously to the citrus melanose severity in an orchard, whereas the ascospores were carried by the wind for a long distance spread. Under high humidity and warm climatic conditions (at 25 ℃ ), the young leaves, shoots and young fruits (within 12 weeks after flowering) were seriously infected by D. citri, however the mature citrus tissues were more resistant to this pathogen attack. Therefore, these phenological periods of citrus are also a critical stage for the prevention and control of citrus melanose. The copper fungicide can act as a good preventative against citrus melanose, but it is susceptible to rain erosion, and is phytotoxic to citrus plant when it is used in hot weather. The other pesticides such as mancozeb and strobilurin etc., play a good control effect on citrus melanose, but they are also facing the risk of increasing resistance to D. citris, because of long-term use of the same fungicides. Some antagonistic microorganisms such as Burkholderia gladioli, Pseudomonas pudia, P. fluorescens, Bacillus subtilis, B. velezensis, B. amyloliquefaciens, Trichoderma asperellum, and T. asperelloides etc., all play a strong inhibitory effect on the mycelial growth or conidia germination of D. citrus, which could provide a reference for commercial application on management approaches of citrus melanose in the field. The above results indicate that, in recent years, some progresses such as species identification and detection, genetic diversity, genomic information, infection cycle, pathogenic mechanism, occurrence patterns, prevention and control measures of D. citris have been made by many researchers at home and abroad, but the following issues still need to be further explored. (1) Whether the D. citris forms a special infection structure to successfully penetrate the leaves and peels of citrus with a waxy layer. (2) The pectinase secreted by D. citris is an important virulence factor, but the types, encoding genes and functions of pectinase still need to be clarified. In addition, the presence of other important virulence factors such as toxins, effector proteins etc., needs to be further analyzed. (3) The propagules of D. citris only form on dead wood, but not on non-dead branches, so the molecular regulation mechanism of asexual spores and ascospores development needs to be studied in D. citris. (4) The community of antagonistic microorganisms increases in the citrus leaves, when they are attacked by D. citris. So, how the citrus plant recognizes the molecular signals of D. citris to regulate autoimmunity, and the molecular interactions between citrus and D. citris remain to be understood. In conclusion, an in-depth understanding of the infection structure, virulence factors, molecular mechanisms of sporogenesis of D. citris, and the molecular signaling pathway of the recognition of D. citrisby host will help to provide resources for citrus disease resistance-breeding, and also provide new targets for accelerating the development and application of fungicides for the prevention and control of citrus melanose. At the same time, the population of D. citris is abundant, and it is a heterogeneous fungus, with frequent sexual reproduction. So it is necessary to strengthen the monitoring of its sensitivity to pesticides such as mancozeb, etc.. And these measurements such as mixing pesticides scientifically and reasonably, using biocontrol agents and plant resistance inducers, will reduce the case of fungicide resistance against D. citris, and improve the comprehensive prevention and control ability of citrus melanose.