Elsinochrome phytotoxin production and pathogenicity of Elsinoë arachidis isolates in China

Peanut scab caused by Elsinoë arachidis is found throughout China’s peanut-growing areas. Elsinochrome produced by E. arachidis is a perylenequinone photosensitive mycotoxin vital to the pathogenic process of the pathogen. In this study, the complex mechanism underlying the regulation of elsinochrome biosynthesis by E. arachidis was investigated based on various nutritional and environmental factors. The initiation of elsinochrome biosynthesis depends on light. E. arachidis produced substantially more quantities of elsinochrome when grown on a semi-synthetic medium (PDA) than when grown on synthetic media with defined ingredients in the presence of light. Elsinochrome accumulation decreased when adjusted with either citrate or phosphate buffers and changing pH suppressed the radical growth. At temperatures ranging from 10°C to 25°C, the production of elsinochrome increased, peaking at 28°C, and it decreased slightly at 30°C. 63 field-collected isolates from China were assessed for the level of elsinochrome production, and pathogenicity analysis was conducted by selecting 12 strains from each 3 of the 4 groups with different levels of elsinochrome production. A direct correlation was observed between elsinochrome production and pathogenicity among the isolates. The results showed elsinochrome biosynthesis to be controlled by E. arachidis and showed elsinochrome to be a vital virulence factor of E. arachidis, required for disease severity.

Elsinochrome has been reported to induce necrotic lesions on citrus leaves, causing electrolyte leakage from citrus cells and toxicity to tobacco cells [13]. This effect is primarily attributed to the high yield of singlet oxygen and superoxide. Disrupted Efpks1 in E. fawcettii completely abolished the production of elsinochrome, and the ability to develop lesions on citrus was significantly reduced, suggesting that elsinochrome produced by E. fawcettii fungi at full virulence [12][13][14].
Secondary metabolite biosynthesis by microbes can respond to environmental and nutritional factors [15]. Understanding the biosynthetic pathway of secondary metabolite toxins can help researchers study the role they play in disease [16]. According to previous studies, the accumulation of cercosporin was affected by complex factors, during which light serves as critical condition [17]. The production of elsinochrome by E. fawcettii has been proven to rely on light and pH as vital factors. However, the regulation of elsinochrome production by E. arachidis in response to environmental conditions and the diversity of elsinochrome production and fungal virulence by different E. arachidis isolates in China remains unclear.
For this reason, the objective of the present study was to investigate the effect of media, temperature, light, pH and cultivation time on biosynthesis of elsinochrome and growth of E. arachidis to determine whether isolates from different China's peanut-producing areas produce elsinochrome and determine the relationship between this ability and fungal virulence.

Strain and culture condition
E. arachidis strains used in this study were isolated from different peanut-growing areas in China (S1 Table). All strains were sub-cultured for purification by single spore and cultured on PDA under continuous light condition (5 microeinstein (μE) m -2 s -1 ). For the preparation of fungal inoculum, 10-day-old mycelium was suspended in sterile water, and then the concentration was adjusted (OD 600 = 2.0) (Zhao et al. 2017). Mycelium suspension (3ul) was placed on the surface of PDA (15ml) plate (90mm diameter). The colony diameter was measured after 4 weeks [5].

Toxin extraction and quantitative analysis
Plates were incubated at 25˚C under continuous fluorescent light. Colony diameters after inoculation were measured at 5, 10, 15, 20, 25, 30 and 35days in two perpendicular cross sections, respectively. For elsinochrome quantification, the method of Liao was referenced with slight modification [13]. 20 agar plugs (5mm diameter) were cut and extracted twice with acetone in the dark. Subsequently, the absorbance was measured at 468nm with spectrophotometer. Elsinochrome concentration was calculated using the standard curve method [5]. There were 3 replicate plates for each treatment.

Pathogenicity test
Pathogenicity of E. arachidis isolates was tested by inoculating mycelium suspension using the method of Zhao [18]. Baisha1016 susceptible to E. arachidis served as a host [1]. Mycelium suspension was sprayed onto the peanut leaves, and then incubated in a chamber under constant light condition at 25˚C for lesion formation [19]. The disease severity was calculated by Fang with some modification [2]: Disease index = (0.1n + 0.2n + 0.4n + 0.6n + 0.8n + 1.0n) / N ×100 n denotes the number of branches at each level; N is the total number of branches investigated

Effect of environmental factors on growth and accumulation of elsinochrome
The radial growth rate of the E. arachidis was slow over the first 5d. Yet the growth rate increased rapidly in the subsequent 10-20d, colony diameter reached a maximum at 25d. The accumulation of elsinochrome generally increased as culture time continued, and then stabilized at 30d. Therefore, maximum toxin production was detected at 30d (Fig 1A).
Growing isolate on OA, PDA, and PSA led to precocious elsinochrome production in the presence of light, however, the accumulation of elsinochrome decreased 10-fold when the isolate was grown on CM. Interestingly, the production of elsinochrome was undetectable on MM, the composition of MM was the same as that of CM, only yeast extract and casein hydrolysate. MM cannot support fungal growth and produce less elsinochrome in MM than CM, suggesting that casein hydrolysate and yeast extract can support fungal radial growth and maximal elsinochrome production. The largest elsinochrome production was found on OA, and PSA was considered the optimum growth medium (Fig 1B).
Mycelial growth and elsinochrome production of E. arachidis isolate were significantly affected by light. Elsinochrome production was greatest in the presence of light, and decreased sharply when E. arachidis was placed under 12h photoperiod conditions, completely absent in the dark. In addition, dark conditions were most suitable for colony growth (Fig 1C).
Colony growth increased as the temperature rose from 10˚C to 25˚C. It peaked at 28˚C and was slightly and rapidly diminished at 30˚C. It did not grow at 5˚C or 35˚C. The range of temperature 25-28˚C contributed to elsinochrome production ( Fig 1E).
The fungus can grow within the pH range of 3.1-7.6 with citrate or phosphate buffers. The optimum pH was found in unbuffered PDA. Colony diameter of the isolate and elsinochrome accumulation both decreased when the fungus was grown on alkaline or acidic medium (Fig 1F).

Pathogenicity test
A total of 12 isolates cultured from 4 different elsinochrome groups were assessed for pathogenicity on peanut leaves and production of necrotic lesions. LNXC-A05 was highly virulent to peanut, LNSY-A01 exhibited reduced virulence, LNJH-C01 was moderately virulent, and the disease index was 41.7, 10.0, and 20.97, respectively (Fig 3). Examination of the correlation between disease index and the yield of elsinochrome accumulation in culture showed Pearson  correlation coefficient r = 0.964, P = 4.196 × 10 −7 , suggesting a direct correspondence between the accumulation of elsinochrome and pathogenicity (Fig 3). As stated above, elsinochrome is considered a vital virulence factor of peanut scab.

Discussion
Elsinoë species are common phytopathogens causing scab and spot on field crops (e.g. cassava, bean, peanut and ornamentals) and economic crops (e.g. poinsettias, avocado, mango, grape and citrus) [3]. Many Elsinoë can produce elsinochrome [6][7]. Elsinochrome is a light-activated, nonhost-selective phytotoxin that can damage cell membranes and induce electrolyte leakage. Recent studies based on molecular and genetic tools verified the critical role of elsinochrome in lesion development [20]. The ability to produce elsinochrome may serve be an important means by which Elsinoë can infect different crop species and cause disease.
The production and accumulation of elsinochrome showed stable increases over culture time under laboratory conditions, and these increases were affected by light, temperature, and pH. Here, light was found to be the most indispensable signal for elsinochrome biosynthesis in E. arachidis; elsinochrome production was completely abolished when the organism was kept in darkness. In E. fawcettii, although the biosynthesis of elsinochrome was suppressed in darkness, there was still a small amount of synthesis. The ambient pH was found to be one of the most important signals suitable for use as a regulatory cue for secondary metabolites and developmental processes in many organisms [21][22]. LNJH-C01 produces large quantities of elsinochrome when grown at PDA and it suppressed radical growth across different pH values in the presence of citrate and phosphate. However, the quantities of elsinochrome in E. fawcettii were largest when it was grown under alkaline conditions on PDA which indicated the complexity of metabolic pathway, even the same substance is synthesized differently in different species. In general, elsinochrome biosynthesis of Elsinoë. spp was complex and showed differences between species.
A wide range of levels of elsinochrome accumulation was observed among isolates of E. arachidis from different parts of China. In the previous study, elsinochrome, which induced necrotic lesions on peanut leaves, was demonstrated to be an important virulence factor to E. arachidis [4]. To assess the correlation between the production of elsinochrome and the pathogenicity of pathogens, we selected 12 strains for pathogenicity assays and grouped them by differences in toxin production. Pearson correlation coefficient was used to analyze the relationship between pathogenicity and elsinochrome accumulation, r = 0.964, suggesting that among E. arachidis isolates the accumulation of elsinochrome in PDA and the pathogenicity had a direct correspondence. However, the manner by which elsinochrome causes virulence and what the role elsinochrome may play in the mechanism underlying pathogenesis of E. arachidis needs further study.
In general, a complex, interrelated regulatory network causes to the accumulation of elsinochrome in E. arachidis. Direct correspondence between pathogenicity and elsinochrome accumulation here support our previous finding that elsinochrome is necessary to fungal virulence.
Supporting information S1 Raw data. Mycelial growth and the production of elsinochrome of LNJH-C01 under different enviromantal conditions. (XLS) S1 Table. The information of 63 isolates of E. arachidis. (XLS)