Several citrus diseases are currently documented in China and around the world. The generally occurring fungal diseases include melanose, gummosis, and stem-end rot caused by Diaporthe spp.; branch cankers caused by Botryosphaeriaceae ^[1][2][1,2]; scab caused by Elsinoë spp. ^{[3][4][5][6][7][8][9]}[3,4,5,6,7,8,9]; black rot caused by Alternaria spp. ^{[10][11][12][13][14]}[10,11,12,13,14]; greasy leaf spot caused by Cercosporoid genus ^[15][16][15,16]; anthracnose caused by Colletotrichum spp. ^{[17][18][19][20][21][22][23][24][25]}[17,18,19,20,21,22,23,24,25]; and blue and green mold caused by Penicillium spp. ^[26][27][28][26,27,28]. Among these fungal diseases, melanose, gummosis, and stem-end rot caused by Diaporthe spp. have a significant impact on citrus production ^[29][30][29,30]. At the same time, some Diaporthe spp. have also been reported as endophytes and/or saprobes on citrus ^{[29][30][31][32][33][34][35][36][37]}[29,30,31,32,33,34,35,36,37].

Melanose disease was not a major problem in citrus crops prior to the 1990s. However, the accumulation of a large number of dead branches or trees results in an increase in fungal inocula in old citrus orchards worldwide. Currently, melanose has become the major fungal disease of citrus in China, dramatically reducing the commercial value of citrus fruits (Figure 1). Diaporthe spp., have been isolated from citrus hosts in many citrus-growing regions of China, e.g., Jiangxi, Zhejiang, Guangxi, Guangdong, Shaanxi, Fujian, Hunan, Chongqing, Yunnan, etc.

Previous studies about Diaporthe spp. have largely concentrated on species identification, especially the species associated with specific hosts. The molecular taxonomy of the genus Diaporthe related to citrus and allied taxa has made great advances in recent years. The phylogenies based on multiple loci provide a more robust and comprehensible taxonomy and nomenclature for D. citri and will serve as a starting point for field study by plant pathologists, breeders, and mycologists. Such information may be used to improve disease management and the deployment of citrus cultivars with species-specific and/or broad-spectrum resistance.

All citrus species, including grapefruit, clementine, lemon, lime, mandarin, orange, satsuma, and tangerine, are susceptible to melanose. Phomopsis citri was first recorded as a citrus parasitic fungus causing stem-end rot symptoms in Florida, USA [38]. Its teleomorph (sexual stage) is D. citri [39]. In addition to D. citri, many other Diaporthe species were also detected in citrus hosts. They could be pathogens, endophytes, or saprobes on citrus ^{[29][31][40][41][42][43][44][45]}[29,31,40,41,42,43,44,45].

Citrus melanose is caused by D. citri, which belongs to Kingdom Fungi; Ascomycota; Sordariomycetes; Diaporthales; Diaporthaceae; Diaporthe ^{[46][47][48][49][50][51][52][53]}[57,58,59,60,61,62,63,64]. The genus of Diaporthe was established by Nitschke ^[54][65]. Phomopsis is the anamorphic (asexual stage) name of Diaporthe ^{[38][52][55][56][57][58][59]}[38,63,66,67,68,69,70]. The genus Diaporthe shows high species diversity; more than 1200 species named “Diaporthe” and about 1050 species named “Phomopsis” have been recorded in MycoBank lists (http://www.mycobank.org; accessed on 9 June 2021).

2. Epidemiology, Life Cycle, and Symptomatology

Citrus melanose is caused by D. citri, which attacks foliage, fruits, and twigs when they are immature. Since mature tissues are more immune to pathogen attack, the first 8 to 9 weeks of the citrus growing season are the most vulnerable to pathogen attack. Melanose signs can differ depending on the severity of the infection. At the end of the susceptibility cycle, the flyspeck melanose symptoms appear ^[60][61][130,131]. The fungal inocula can be scattered over a wide range since ascospores are released forcefully and can be spread over a long distance. D. citri is primarily a saprophyte that feeds on and receives its nutrition from dead wood ^[44][62][44,132]. Perithecia and pycnidia are only found on dead and dying twigs and fruits showing stem-end rot. The conidia provided by pycnidia are the primary source of inoculum ^[63][133]. Ascospores are ejected forcibly and play a significant role in long-distance dispersal ^[62][132]. As a result of the widespread dissemination of a vast number of ascospores, the number of cases of infection is rising ^[64][134]. When ascospores or conidia of Diaporthe land on the surface of a plant, the disease will be triggered. Pathogens thrive in dry environments with temperatures ranging from 17 to 35 °C [44]. The germination of spores requires approximately 10 to 24 h of moisture, depending on the temperature ^[44][64][44,134], and the germination and formation of a germ tube takes 36 to 48 h ^[63][133]. After that, the citrus melanose pathogen directly penetrates the cuticle layer tissue and infects the plant. D. citri could overwinter on debris, e.g., mummy fruits, dead stems, branches, and dry leaves. Perithecia could form on debris next year. Ascospores are produced in proportion to the amount of dead wood present in a canopy. These spores contribute slightly to the disease severity of an orchard, but they are carried by the wind and spread across long distances. Conidia, developed in mature pycnidia, can continuously infect citrus during the growing season. Conidia can be dispersed to nearby citrus trees with raindrops, which most probably cause the majority of fruit infections. Nevertheless, conidia can also be transmitted through the air over long distances when rainfall is scarce. Symptoms appear as discrete small, sunken, brown spots about one week after infection, which later become raised and filled with reddish-brown gum. The leaf pustules are initially surrounded by a yellow halo. Diseased areas regreen later and create corky pustules. On fruits, pustules can grow relatively large and can crack, creating a pattern of mudcake. The severity of the disease is determined mainly by the amount of inoculum-bearing dead wood in the canopy of the tree and the duration of the wetting period following rainfall or sprinkler irrigation. Wet, rainy conditions, especially when rain showers occur late in the day, and fruits staying continuously wet on warm nights are conducive to infection.

3. Main Management Approaches of Melanose Disease

The yield is almost unaffected by melanose disease, and the juice processing is unaffected as well. However, the quality of the fruit for marketing and exportation suffers the consequences. In order to avoid poor quality and fruit deterioration caused by citrus melanose, integrated management practices should be implemented. Integrated pest management (IPM) is now largely recognized as the most effective way to protect plants. Its ultimate objective will be to maintain pest populations below economically injurious levels without or just with minimal pesticides. Although IPM must rely on pesticides currently, minimizing chemical inputs while maintaining crop quality at an economically viable level is a basic requirement for plant protection. To achieve this goal, it is critical to understand the disease epidemiology at various points in time while performing pest control ^[62][65][132,136]. Currently, no resistance cultivars are available for melanose control in practice. The removal of dead wood to reduce the pressure of melanose fungus is both time-consuming and labor-intensive. Nevertheless, pruning dead branches should be performed on a regular basis. Proper pruning enhances air circulation within the canopy of the plant, keeping it dry and reducing opportunities for pathogens to survive and cause infections. It will also improve the effectiveness of fungicide infiltration into the foliage ^[43][66][43,137]. Furthermore, avoid planting sensitive citrus cultivars or species in high-rainfall zones, such as sweet orange, grapefruit, and pumelo ^[66][67][137,138]. Other management practices, such as citrus plantations in low-rainfall and sunny zones, should be implemented. Interplanting citrus with non-susceptible hosts is also a feasible measure ^[66][68][137,139].

3.1. Chemical Control

Application of fungicides is still the most commonly used method to control melanose disease on citrus. Many fungicides have been tested for melanose control. Copper is a protective compound, which forms a layer on the surface of plant tissue, e.g., fruit, protecting it from infection. The gap in the protective copper layer, however, grows larger as the fruit grows and expands. If conditions are favorable for the pathogen infection, the copper layer needs to be renewed through another spray. The melanose fungus stored in dead wood is slightly affected by copper spraying. The use of copper fungicides before flowering will not reduce infection. A copper fungicide must be applied on the fruit surface to provide efficient melanose control. In the case of serious infection in late summer, additional protectant spray should be applied ^[69][140]. Applications of pyraclostrobin to the spring flush growth of citrus trees are much more efficient for controlling melanose, scab, and Alternaria brown spot than those of famoxadone or copper hydroxide ^{[44][70][71][72][73]}[44,141,142,143,144]. Bushong and Timmer ^[74][145] demonstrated that azoxystrobin was a highly effective preventative spray for melanose, whereas benomyl and fenbuconazole were not. As post-infection treatments for melanose, none of the fungicides are successful. In Japan, dithianon and mancozeb were used to spray alternately from June to August to control this disease ^[75][146]. In Pakistan, five chemicals were tested at recommended doses, including penflufen, copper hydroxide, tebuconazole plus trifloxystrobin, and difenoconazole, for controlling melanose disease. When used as a protectant, copper hydroxide was found to be the most effective for the management of citrus melanose ^[76][147]. Whereas Anwar et al. ^[77][148] evaluated six different fungicides for citrus melanose control, the use of mancozeb led to a significant inhibition of fungal growth. Similarly, several chemicals, including mancozeb and fenbuconazole, were found to be effective in controlling citrus melanose in China and other countries ^{[40][43][77][78][79]}[40,43,148,149,150].

3.2. Biological Control

Although chemical control plays an important role in managing plant diseases, overuse of chemical pesticides has raised severe issues about food contamination, environmental pollution, and phytotoxicity. Biocontrol is a viable option as it is friendly to the environment. Biological control of plant diseases with antagonistic bacteria is a viable alternative to chemical control. Many antagonistic bacteria are known to play important roles in the sustainability of natural ecosystems, and some of them can be employed as inoculants to stimulate plant growth and resistance. For melanose control, more and more biocontrol candidates have been developed, e.g., Burkholderia gladioli: TRH423-3, MRL408-3, Pseudomonas pudia: THJ609-3, and P. fluorescens: TRH415-2, and selected for their antifungal effectiveness against D. citri using dual-culture testing. Disease suppression was observed after pretreatment with the rhizobacterial strains, with varying degrees of protection rates for each rhizobacterial strain. Following the pathogen inoculation, subsequent treatment with the rhizobacterial strains also enhanced protection rates. The rhizobacterial strains might be especially useful in organic citrus production where chemicals are strictly forbidden ^[80][151]. Similarly, pre-treatment with P. putida strain THJ609-3 resulted in a decreased disease incidence. When the infection behaviors of D. citri and necrosis deposits on plant tissues were examined using a fluorescent microscope, it was shown that the process of disease development was reduced after being treated with the bacterial strain, especially the conidia germination rates, which were significantly lower after being pretreated with the strain THJ609-3. Furthermore, morphological abnormalities of the germ tubes were also observed. These results pointed to the bacterial-direct antifungal action on the leaf surfaces as a potential cause of disease reduction ^[81][152]. Thiobacillus species were used to generate bio-sulfur, which was investigated as an alternative to managing citrus melanose. It was found that melanose disease severity was lower on bio-sulfur pretreated citrus leaves than on untreated leaves, suggesting that bio-sulfur might be applied as an environmentally friendly alternative to control citrus melanose ^[82][153]. Bacillus velezensis CE 100, an effective biocontrol agent, has been used to control D. citri. In dual culture plates, D. citri mycelial growth was significantly suppressed by strain CE 100, suggesting that some volatile substances inhibited the growth of D. citri. It was also observed that the bacterial culture filtrate (BCF) of strain CE 100 inhibited D. citri growth. Microscopic examination indicated that BCF had a substantial impact on the pathogen hyphal shape, most probably the result of numerous cell-wall disintegrating enzymes and metabolites generated by strain CE 100. Interestingly, D. citri conidial germination was decreased by approximately 80% when 50% BCF of strain CE 100 was used ^[83][154].