Growth characteristics and the host range of the pathogen causing black spots on the Citrus maxima

Abstract:【Objective】Citrus maxima‘Guanximiyou'is an important fruit tree grown in China, especiallyin Fujian province, and is rich in medicinal and nutritional values. The pomelos black spot, caused by Phyllosticta citriasiana, which occurs on the leaves and fruit, is an important pomelo pathogen leading topremature fruit drop, yield losses, high control costs, and is common throughout East Asia. It has becomeone of the major constraints in the production of high quality wholesome pomelo fruits. Mycelia and conidia of the P. citriasiana serve as primary inocula of fruits and leaves of the C. maxima ‘Guanximiyou'. Theconidia of the Phyllosticta attachment to the hydrophobic surface is important to trigger germination. Butlittle is documented about the conidia germination of the P. citriasiana on the hydrophobic surface and itshost range. Growth characteristics and the host range of P. citriasiana infecting pomelos were determinedin this study to ensure better understanding of the occurrence rules of this disease and to help design anintegrated disease control strategy.【Methods】The 1.5 mL-microcentrifuge polypropylene tubes serve asthe hydrophobic surface to test conidial germination with different solutions and incubation times. 50-μLdroplets of P. citriasiana conidial suspension (4×105P. citriasiana conidia/m L) in different solutions(0.7mol·L^-1 Na Cl solution, 1% glucose solution, 10% glucose solution, and water, respectively) were placed inthe tubes and incubated at 25 ℃ for 24, 48, and 72 h, respectively. The percentages of germinated conidiawere determined by examining 50 conidia on each drop by using light microscopes(Olympus CX41-RF).On the other hand, the effects of different media, temperature, and illuminance on the mycelial growth of P. citriasiana were also investigated. To determine the effect of media on mycelial growth, 2-3 mm diame-ter mycelial blocks were cut from the margins of 10-day-old cultures of P. citriasiana and placed respec-tively in different media plates and incubated at 25 ℃. After 4, 7, 9 days of incubation, colony diameterswere measured. Similarly, influences of temperature(10, 15, 20, 25, 30, 35, and 40 ℃) and illuminance(0, 450 and 1 980 lx, 24 h·d^-1) on the mycelial growth were conducted as described in the above method.Lethal temperature of conidia and mycelia was determined by testing their viabilities when the conidia ormycelia suspension incubated in hot-water(45, 50, 55, and 60 ℃) for 15 min, respectively. In addition,the leaves or fruits of 28 Citrus plants were inoculated with mycelial pellets of P. citriasiana using awounded inoculation technique to explore the host range of P. citriasiana in vitro.【Results】Conidial germi-nation on the hydrophobic surface(1.5 mL-microcentrifuge tube) was influenced significantly by the ger-mination solution and incubation time. P. citriasiana showed higher levels of conidial germination in water(52.7%) and 1% glucose solution(61.4%) after 48 h at 25 ℃, whereas there was<30% germination in wa-ter(8.3%) and 1% glucose solution(25.3%) after 24 h at 25 ℃. The germination rate was reduced in wateror 1% glucose solution after a longer incubation time(72 h). In addition, there was no significant differ-ence in the conidial germination in 10% glucose solution from 24 to 72 h, with maximum(39.1%) after 48 h at 25 ℃, while the conidial germination rate was very low in 0.7 mol·L- 1Na Cl solution, and peaked(16.4%) after 72 h at 25 ℃. The mycelial growth was obviously affected by the different media. Oatmealagar(OA), potato sucrose agar(PSA), and complete medium(CM) were relatively favorable media for P. citriasiana mycelial growth. Moreover, the mycelial growth rate(9 d) was significantly enhanced by adding2% sorbi-ny. By contrast, mycelia grew slowly on the 20% sucrose complete medium(SCM), 0.6 mol·L-1sorbitol casamino acid agar(0.6 mol·L-1-SCAA), 0.1 mol·L^-1 sorbitol casamino acid agar(0.1 mol·L-1-SCAA), and yeast extract casamino acid sucrose agar(YECASA). The mycelial growth was sensitive to thetemperature. The condition of temperature at 25-30 ℃ was best for mycelia growth, but the mycelial growthrate was significantly reduced or completely inhibited under temperatures at ≤20 ℃ or ≥35 ℃. And the my-celial growth rate was not significantly different under illuminance at 0, 450, 1 980 lx(24 h·d-1). In addi-tion, the mycelia or conidia treated by a water bath at 55 ℃ for 15 min were not viable, whereas some ofthem could survive when they were treated by a water bath at ≤50 ℃ for 15 min. On the other hand, themycelia could infect wounded leaves or fruits of pomelos such as C. maxima‘Guanximiyou', C. maxima‘Longyou 1', C. maxima‘Longyou 2', C. maxima‘Pingshanyou', and lemons such as Ctrus limon‘Femminello Baihua 2', C. limon‘Wufeng 2'and C. limon‘Hainanyaoyong', and Citrus paradisi Macf.in vitro. Moreover, C. maxima‘Longyou 1', C. limon‘Wufeng 2'and C. limon‘Hainanyaoyong'werethe most susceptible to P. citriasiana.【Conclusion】This work significantly contributes to our understand-ing of the rules of conidial germination and mycelial growth, and to our knowledge, this is the first timeanyone analyzed the host range of P. citriasiana. This study indicates that P. citriasiana has some poten-tial hosts that were not previously known, but further field investigations are necessary for host range determination. Furthermore, the influences of other environmental factors such as light, humidity, and tem-perature should not be ignored due to the important roles they could play in conidial germination and in-fection.