Fungal root rot is a common disease in alfalfa crops in several areas of the world, including the USA, Canada, Australia, Iran and China [1
]. Root rot induces discoloration of the infected tissues and subsequent development of black and necrotic areas, often leading to alfalfa plant death. A variety of plant pathogenic fungi has been reported to induce root rot in alfalfa, such as Fusarium
], Mycoleptodiscus terrestris
], Microdochium tabacinum
], Rhizoctonia solani
] and Phoma sclerotioides
]. Paraphoma radicina
was reported to be a new pathogen causing alfalfa root rot in Inner Mongolia, China, and pathogenicity experiments showed that P. radicina
is significantly detrimental to alfalfa growth [15
is the type species of the genus Paraphoma
, although it was originally named Phoma radicina
]. The fungus was initially described in isolates obtained from cysts of Heterodera glycines
in North Carolina, USA, in soybean field soils [17
]. Subsequently, it was also recorded on H. glycines
in Shenyang, China [18
]. Paraphoma radicina
was previously reported as a saprophytic species on the roots of herbaceous and woody plants [16
]. However, in a previous publication, we showed this species to be able to infect alfalfa roots and cause severe root rot [15
]. Pycnidia of P. radicina
are commonly present on black necrotic root tissue of diseased alfalfa. Pycnidia formed on the diseased plant tissue were found to contain many viable conidia, which are believed to serve as inocula in alfalfa infection. Since the teleomorph of the fungus is still unknown [19
], it is considered that conidia play an important role in the spread of the disease and, therefore, conidial germination may be an important factor for successful infection.
Conidial germination, sporulation, colonization and mycelial growth of fungi are greatly influenced by nutritional and environmental factors [20
]. However, information on the influence of these factors on the biological and physiological characteristics of P. radicina
is currently limited. The effects of temperature and pH on mycelial growth have been previously evaluated [15
], where it was found difficult to induce conidiation of P. radicina
in vitro, which may limit further studies on effective disease control. In the present study, our objectives were to determine the effect of (i) culture media on mycelial growth and conidial production, (ii) carbon and nitrogen sources on growth and (iii) temperature and pH on conidial germination, and to determine the lethal temperature of P. radicina
. The outcome of the research described here will contribute to a greater understanding of the etiological agent of the alfalfa root rot epidemic to guide strategies for preventing the spread of this disease and mitigate potential crop yield losses in China.
2. Materials and Methods
2.1. Biological Material
Isolates of P. radicina
were obtained from root tissues of diseased alfalfa plants in Chifeng city, Inner Mongolia, China. The type isolate of P. radicina
(LYZ187) used in all experiments was that previously obtained [15
]. Pure cultures of P. radicina
were grown on potato dextrose agar (PDA) at 25 °C and L:D 0:24 h for four weeks for later use as inocula. Mycelial plugs (5 mm diameter) were aseptically cut from the margins of actively growing colonies with a sterile cork borer and transferred to a new PDA Petri dish. Plates were inoculated by placing plugs face down on each dish, with five replicates per treatment. The dishes were sealed with laboratory film (ParafilmTM
, Oshkosh, WI, USA) and randomly arranged in a 25 °C incubator.
2.2. Mycelial Growth and Sporulation of P. Radicina on Different Agar Media
The growth of P. radicina
was assessed on eight different media (Table 1
). All agar media were autoclaved at 121 °C for 20 min prior to being poured into individual Petri dishes. Colony shape and color were observed in the first week either by the eye or under a dissecting microscope. Photographs were taken with a Handheld Canon DS126391 camera and colony features were recorded. Colony diameters were measured weekly for four weeks, and growth rates were calculated as the colony diameter with less than five millimeters of mycelial plugs. After one week of incubation, five milliliters of sterilized distilled water were added to each plate, and the colony surface was gently scraped with a sterilized glass spreader to dislodge conidia. The resulting suspension was filtered through sterile gauze, and the procedure was repeated with another five milliliters of water. The combined conidial suspension volume was measured, and the concentration of conidiospores was estimated with a hemacytometer. The collection of conidiospores was repeated after four weeks of incubation [23
2.3. Effects of Carbon and Nitrogen Sources on Growth
For carbon source suitability experiments, nine carbon compounds were assessed for their ability to support mycelial growth. The basal medium consisted of 20 g of dextrose, 5 g of KNO3
, 2 g of Na3
, 1 g of MgSO4
, 17 g of agar and up to 1000 mL of distilled water; the other eight carbon sources were sucrose, mannitol, fructose, D-xylose, lactose, soluble starch, maltose and cellulose. Dextrose in the basal medium was replaced with 20 g of each carbon source to prepare individual formulations to be tested [26
]. Growth rates, measured as the colony diameter, were monitored for four weeks.
Seven nitrogen compounds were assessed for their ability to support mycelial growth: Peptone, urea, ammonium nitrate, ammonium chloride, glycine, sodium nitrate and ammonium sulfate. Five grams of each nitrogen source were added to the basal media (20 g of dextrose, 2 g of Na3PO4, 1 g of MgSO4, 17 g of agar and up to 1000 mL of distilled water) to prepare individual formulations to be tested. Inoculated media were incubated in the dark at 25 °C.
2.4. Effect of Temperature and pH on Conidial Germination
To determine the optimal temperature for conidial germination of P. radicina
, conidial suspensions in sterile distilled water were adjusted to 1.0 × 106
spores/mL with a hemacytometer. Using the slide technique [27
], a 10 μL drop of a conidial suspension was placed in a cavity slide using a micropipette. The slide was then placed onto the well of a 90 mm plastic Petri plate on top of a moist filter paper to maintain the humidity level. The plates were sealed with parafilm and incubated (L:D 0:24 h) at 4, 10, 15, 20, 25, 28, 30 or 35 °C, respectively. Treatments were replicated three times. Conidial germination was monitored every four hours through microscopic observation at ×400 magnification using an Olympus CX31 microscope (Olympus, Tokyo, Japan). Samples were observed continuously for 38 h until there were no new conidia germinating. The conidial germination rate was determined by counting conidia in 30 randomly selected fields on each plate. A conidium was considered germinated if the germ tube was at least one-half the length of the conidium.
To assess the effect of pH on conidial germination, sterile water was adjusted to pH 4, 5, 6, 7, 8, 9 or 10 by the addition of 0.1 M NaOH or HCl prior to the preparation of conidial suspensions. These suspensions were allowed to germinate while being kept moist using the slide technique described above. All Petri dishes were incubated at 25 °C in the dark. Observation and evaluation of the conidial germination were carried out as described above.
2.5. Lethal Temperature of Mycelium and Conidia
Mycelial plugs (5 mm diameter) were placed in a sterilized test tube, and then the tubes were placed in a thermostat water bath at 40–55 °C (temperature gradient: 1 °C) for 10 min (preheating: One minute). After cooling, the plugs were taken out and placed face down on PDA media dishes. All plates were incubated for one week at 25 °C in the dark to evaluate mycelial growth capability.
To determine the lethal temperature for conidia, 2 mL of a conidial suspension was taken into sterilized test tubes and subjected to heat treatment as described above. Conidial viability was determined by measuring the germination rate after 34 h in cavity slides as described [28
2.6. Data Analysis
Growth rate, sporulation and conidial germination rate data were subjected to analysis using SPSS Statistics 21 analysis software (SPSS Inc., Chicago, IL, USA) using descriptive statistics, one-way ANOVA and the Duncan test with p ≤ 0.05 as the significance threshold. The effects of carbon and nitrogen sources on growth were statistically analyzed by the least significant difference (LSD) and Duncan tests (p ≤ 0.05).
The establishment of a successful parasitic relationship between fungal pathogens and their hosts is influenced to varying degrees by environmental conditions [20
]. Therefore, the elucidation of the factors influencing fungal growth, sporulation and conidial germination is of practical importance to devise better disease control strategies.
In earlier research, the aerial mycelium of P. radicina
was described as pale olivaceous on OA medium and the colony color as pale luteous due to the production of a diffusible pigment [16
]. Our initial results after evaluating the colony characteristics of P. radicina
on various media agreed with those previously reported. In the present study, we evaluated the effects of eight media on conidia production of P. radicina
and demonstrated that it was able to produce pycnidia and conidia only on PDA and ARDA media, while few conidia were generated on PDA. This is in agreement with earlier studies where it was also reported that pycnidia and conidia of P. radicina
were not produced abundantly on the PDA medium [17
]. Furthermore, in this study, we found that conidia of P. radicina
were not produced on OA. Conversely, previous authors mentioned that the fungus produced abundant pycnidia on OA [16
]. This discrepancy is likely due to differences in culture conditions. Our research showed that P. radicina
displayed the greatest growth rate and sporulation on the ARDA medium, indicating that ARDA is the optimal medium for P. radicina
This research also showed that P. radicina
was able to use all tested carbon and nitrogen sources. After four weeks of incubation, the fungus showed significantly that the most vigorous growth was attained when mannitol was used as a carbon source, and when peptone was used as a nitrogen source. These attributes have been found in other fungal pathogens. For example, the growth rate of Embellisia astragali
was reported to be greatest on mannitol and peptone after four weeks [26
]. External nutrients can promote conidia production in some pathogenic fungi. Thus, Verticillium alfalfa
displayed excellent sporulation on lactose and starch, and the greatest sporulation rate on peptone [30
]. Furthermore, when the carbon nutritional requirements of Phoma medicaginis
were studied, it was found that monosaccharides and disaccharides were nearly equivalent in the production of pycnidia, but superior to polysaccharides [31
]. The same study reported that the formation of pycnidia and conidia was favored on nitrate more than ammonium nitrogen sources in P. medicaginis
. However, we found no conidia production by P. radicina
on any of the tested carbon and nitrogen sources. This may indicate that conditions required for sporulation are more demanding compared to other fungi.
Our research showed that the optimal temperature for conidial germination of P. radicina
was 25 °C. There was little germination at temperatures below 5 °C or above 35 °C. These results were similar to those in earlier reports where other species of fungi from alfalfa were studied [30
]. In comparison, P. radicina
mycelia can grow across a wide range of temperatures from 5 to 35 °C, with an optimal temperature in the range of 25 to 30 °C [15
]. This finding suggests that P. radicina
temperature requirements for conidial germination are in general agreement with those for mycelial growth. Of interest was the result showing that the lethal temperature for mycelial growth was greater than that of the conidia. This may be due to the formation of chlamydospores within the mycelium during incubation.
Fungal conidial germination was found early to be influenced by the hydrogen ion concentration in the growth medium [33
]. In the present study, we observed that P. radicina
conidia germinated at pHs ranging from pH 4 to pH 10, with the optimal at pH 7. In a previous report, we found that the optimal pH for mycelial growth of this fungus was within the range of pH 8 to pH 9, and that the growth was slow when the pH of the culture medium was <pH 4 or >pH 10 [15
]. Despite this difference in the optimal pH for conidial germination versus mycelial growth, these results indicate that both excessive acidity and alkalinity are detrimental to the growth and conidial germination of P. radicina