Effect of interaction between Fusarium solani and Rhizoctonia solani on damping-off and root rot disease of Tetraclinis articulata seedlings

R. solani and F. solani isolates were collected on March 20, 2019 from root rot diseased Tetraclinis articulata seedlings grown in Sidi Amira Forest Nursery (North West of Morocco; 34°02′50″ N, 06°40′34″ W; 130 m elevation). The isolates were stored in a climate chamber at 4°C.

For R. solani inoculum preparation, 250 ml conical flasks, each containing about 10 g of barley grains soaked overnight in sterilized distilled water, were used. The media in flasks were autoclaved for 30 min on two consecutive days. After the flasks were cooled, each was inoculated with a small block (5 mm diameter) taken from the periphery of the seven-day-old cultures on the PSA medium (Potato Sucrose Agar: 200 g potato, 20 g sucrose, 15 g agar-agar, 1000 ml of distilled water). The flasks were then incubated at 27°C for two weeks. During incubation, the flasks were shaken twice a day to ensure the proper growth of fungal mycelium on the barley seeds. 5 g plugs of the fungal-colonized barley seeds were used as the inoculum.

The inoculum of F. solani was prepared after 10 days of culturing the parasite on the PSA medium at a temperature of 25°C. Spore suspension was prepared by scraping the culture in sterile distilled water and then adjusted to 2.10⁶ spore ml⁻¹ using a haemocytometer.

New 400 ml plastic containers with 50 cells (16 cm height and 5 × 5 cm length × width per section) were filled with a (3:1,v,v) mixture of pathogen-free peat and vermiculite up to 15 cm in height and then each container cell was inoculated with one mycelia plug at (50:1,w/w) the growing medium/inoculum of R. solani. A layer (0.5 cm) of peat was added above the inoculum plug. For control, 1 barely meal plug without the inoculum (mock inoculum) was added in each container cell.

Pre-germinated T. articulata seeds (94.4% germination rate) were immersed during 3 hours in the spore suspension of F. solani and shaken every 30 minutes to enhance spore contact with seeds. Control seeds were immersed in sterile distilled water for the same period. All seeds were superficially dried with a sterilized filter paper and transferred to the greenhouse for sowing.

The same day of growing media and seeds inoculation (April 15, 2019), pre-germinated seeds of T. articulata were softly placed in each container cell (2 seeds/cell) and covered with a 0.5 cm layer of the peat-vermiculite mixture in order to fill up the container cell. Four treatments were thus prepared:

Control treatment: uninoculated seeds + uninoculated growing media (4 containers);

The containers with the four treatments were kept in the greenhouse in separated blocks to avoid any risk of contamination during the entire period of the experiment. The breeding stage of the thuja seedlings took place between April 15 and June 17, 2019. The temperature varied from 23 to 28°C and relative humidity ranged between 70 and 95%. The containers were daily watered to saturation at 8h00. No insecticides, fungicides or fertilizers were used and only one seedling was kept in each container cell after thinning.

62 days post-inoculation, the seedlings were removed from container cells, softly washed under tap water and growth parameters (shoot height, primary root and secondary root length, number of leaves, and secondary root number) were recorded for each seedling. A reduction in growth was calculated for each growth parameter using the following formula (Rahman et al., 2016): (1) Reduction in growth (%)= [(G1−G2)/G1]*100, Reduction\,in\,growth\,\left( \% \right) = \,\left[ {\left( {{G_1} - {G_2}} \right)/{G_1}} \right]*100, where G₁ is the mean of the measured parameters for control plants and G₂ is the mean of the measured parameters for inoculated plants.

Disease assessment was made with disease severity categories (Lilja et al., 1992; Khangura et al., 1999; Drizou et al., 2017). For the purpose of this study, the rating scale adopted by El Haddadi et al. (2019) for F. solani damping-off disease assessment and El Haddadi et al. (2020) for R. solani root rot disease assessment on thuja seedlings was used. The condition of the seedlings was classified according to their symptoms as follows: for the hypocotyls disease, the seedlings were categorised on a scale of 0–4 (0 = no lesions or discoloration, 1 = small lesion (<2 mm) on the hypocotyl with reddish to brown coloration affecting <25% of the hypocotyl length (Figure 1), 2 = dark to brown, brown, or black lesions covering 25–50% of the length of the hypocotyl (Figure 2), 3 = necrosis affecting more than 50% of the length of the hypocotyl (Figure 3), 4 = completely dead or not emerged), for the epicotyl disease on a 0–4 scale (0 = no symptoms, 1 = <25% of the needles were yellowish, including twisting of needles and needle-tip dieback, 2 = 25–50% of the needles were yellowish or brown and some stunting may have been observed, 3 = >50% of the needles were brown and the seedlings were dying, 4 = completely dead or not emerged), for the primary root disease on a 0–6 scale (0 = no lesions, 1 = small lesions on primary root, 2 = discoloration or necrosis covering less than 25% of the primary root, 3 = necrosis covering up to 25–50% of the primary root, 4 = necrosis covering 50–75% of the primary root, 5 = necrosis covering >75% of primary roots, 6 = completely dead or not emerged) and for the lateral root disease, on 0–6 (0 = no symptoms, 1 = 1 lateral root girdled, 2 = 2 to 5 lateral roots girdled, 3 = 6 to 9 lateral roots girdled, 4 = 10 to 13 lateral roots girdled, 5 = >14 lateral roots girdled, 6 = dead or not emerged).

Figure 1

Figure 2

Figure 3

Disease index DI (%) (Aoyagi et al., 1998) was calculated as: (2) DI(%)=∑i=1nNi*diN*D*100, DI\left( \% \right) = \sum\limits_{i = 1}^n {{{Ni*di} \over {N*D}}*100,} where Ni: Number of plants in the disease category; di: Numerical value of the disease category; N: Number of plants in all categories; D: Maximum value on the rating scale.

Reisolation of pathogens present in symptomatic seedlings was performed by removing fragments from the edge of a lesion in the roots, hypocotyl and epicotyl. Five pieces (approximately 0.5 x 0.1 cm) were sequentially surface-treated (disinfected with alcohol at 90° for 1 to 2 minutes, rinsed thoroughly with sterile distilled water, dried on a sterile filter paper) and then deposited in Petri dishes containing the PSA medium + Chloramphenicol ≥ 98% (0.05 g/l). Four Petri dishes from each treatment were prepared for this issue. The cultures are incubated in darkness at 25°C. The identification of the fungi was based on morphological criteria using microscopic observations of the culture after purification on different culture media (Parmeter & Whitney, 1970; Nelson et al., 1983). The isolation percentage Pi (%) was obtained by applying the following formula (Chliyeh et al., 2017): (3) Pi(%)=(NX/NT)*100, {\rm{Pi}}\left( \% \right) = \left( {{{\rm{N}}_{\rm{X}}}/{N_{\rm{T}}}} \right)*100, where N_X is the number of segments containing the fungal species X and N_T is the total number of colonies of all isolated species.

Normality was checked based on the Shapiro-Wilk test (P≤0.05). Data were analysed according to the non-parametric Kruskal-Wallis test at 5% probability. Dunn's Multiple Comparison Test was used to separate the means of disease severity, seedling survival, shoot height, leaf number per plant, primary and lateral root number and length. All statistical analyses were performed using XLSTAT software (v2016 02.27444).

Both R. solani and F. solani cause pre-emergence damping-off, post-emergence damping-off and root rot:

R. solani: The symptoms of post-emergence damping-off of thuja seedlings caused by R. solani occur (3–5 weeks) at or slightly below the groundline and result in water-soaked, brownish or blackish lesions that rapidly become sunken or constricted. Lesions can progress along the stem and reach the roots. For root rot, both the primary root and lateral roots are affected, brownish and blackish lesions extend along the root system and form necrosis of the cambium tissues, resulting in its decay. The disease destroys lateral roots and makes them fragmented. Root rot is often associated with the lack of feeders (Figure 4).

Figure 4

F. solani: Newly infected seedlings (3–6 weeks) typically have scattered chlorotic or curled needles followed by tip dieback, wilt symptoms, and stunting as the disease progresses. The fungus causes stem cankers either just above the groundline or higher on the main stem. The disease spreads along the root system. Cankered roots often look black and show a lack of fine root development and exhibit extensive cortical decay so that the epidermis and cortex are easily stripped away from the core tissues (Figure 4).

Pre-emergence damping-off disease was recorded more in containers with the two pathogens (RsFs treatment: 26.93%) than in those with only one pathogen: 21 and 22.5% for Rs and Fs treatments respectively (Table 1). The same behavior was observed for post-emergence damping-off disease; concomitant inoculation (RsFs treatment) caused the melting of 19.06% of seedlings, while mono-inoculations caused only 7.12% (Fs treatment) and 11.12% (Rs treatment) of mortality (Table 1). In short, the damping-off disease reduced survival until 54% for seedlings with RsFs treatment compared to 67.87 and 70.37% for seedlings with Rs and Fs treatment (Table 1). Control seedlings showed a high survival rate (90%) near the germination rate of the seeds lot used in the experiment (94.4%). The combination of the two pathogens resulted in more damping-off disease, increasing mortality rate in the early stage of seedling establishment than F. solani or R. solani alone, and suggesting a synergic effect of F. solani when combined with R. solani.

The aerial system responsible for photosynthesis has been greatly impacted by the action of the two pathogens (Table 2). Whether applied individually or in combination, the impact is visually observed on the appearance of the plants (Figure 5). The average shoot height decreased by 39.2% for Rs, 36.38% for Fs, but only by 28.4% for RsFs (Table 2). Plants inoculated by one pathogen were shorter than plants uninoculated or inoculated by both pathogens. The two pathogens slow down apical growth and cause stunting symptoms to young seedlings. The number of leaves in the aerial system declined by 41.47% for Fs, 13.53% for Rs and 23.09% for RsFs. This means that the impact of F. solani alone is more virulent on the foliar system than with the presence of R. solani. The effect of R. solani on the foliar system can be considered secondary in a broad context. It is likely that the presence of R. solani mitigates the impact of F. solani on apical growth and development of the photosynthetic system.

Figure 5

The number of lateral roots recorded on each seedling (Table 2) showed a significant difference between inoculated (11 lateral roots/plant) and uninoculated seedlings (17 lateral roots/plant), which means a loss of 30 to 33% of the fine root system compared to control plants. Both or alone, the two pathogens showed the same effect on the lateral root number. Lateral roots and primary root length (Table 2) was significantly reduced under different treatments compared to the control treatment. Root rot disease, causing fragmentation of roots, proved more accentuated when the two pathogens acted together. This once again confirms the synergistic interaction of the two pathogens. The total length of the root system measured on infected seedlings (Table 2) also showed the same decreasing trend comparing to control plants (86.32 cm). Seedlings under combined inoculation of RsFs recorded the shortest root system (48.98 cm) with a reduction in growth of 43.26%; nevertheless, multiple comparisons between the effect of RsFs and Rs treatments (p=0.896) on the one side and between RsFs and Fs treatments (p=0.113) on the other side were not statically significant. R. solani and F. solani tend to have a similar impact on the radical system, but the combination of the two pathogens demonstrates significant synergism and reduces the length and density of the radical system, which will have a very negative effect on the absorption of water and nutrients necessary for young seedling establishment.

Disease severity recorded on the epicotyl (Table 3) confirms that R. solani does not directly affect the leaves (epicotyl), but its destructive effect remains very pronounced at the level of the stem (hypocotyl) and the roots. F. solani disease symptoms could be seen in all parts of the infected seedlings and the severity of the disease was similar to that observed with the Rs treatment on the stem and primary root. F. solani is relatively more virulent, when inoculated alone, at the level of lateral roots compared to R solani. However, the combination of the two pathogens causes considerable damage in all parts of seedlings. The disease-index (Table 3) recorded significant differences (p<0.0001) between the RsFs treatment (70.1%) compared to Rs (43.04%) on the one hand, and RsFs (p<0.0001) compared to Fs (56.25%) on the other hand.

R. solani and F. solani were often re-isolated from the same part of diseased plants (stem or root), demonstrating that these pathogens can co-exist in thuja seedlings (Table 4). The isolation percentage Pi (%) of pathogens (fungal recovery) varies depending on the treatment applied (Table 4): it was relatively high for R. solani (50–70%) when applied alone, low when associated with F. solani (13–25%) and with a remarkable absence on epicotyl. Likewise, the isolation percentage of F. solani decreased when the fungi were combined with R. solani. The low isolation percentage of R. solani from the roots and hypocotyl of diseased seedlings inoculated with the mixed inoculum (Table 4) showed this pathogen as a weak competitor to F. solani. However, the elevated mortality (Table 1) and disease severity (Table 3) observed when it is in combination with F. solani indicates it may be important as a primary invader. Although isolation percentages of the two pathogens from the combined treatment (RsFs) were relatively low than those observed in mono-inoculation treatments (Rs and Fs), the mutual action of the two pathogens was more lethal (causing 46% mortality) and more damaging to young seedlings (increasing the disease index to 70.1%) than when they acted individually. This confirms that these two pathogens act in synergy.

Previous studies on different pathosystems have also emphasized a synergistic effect when soil-borne pathogens were combined. R. solani was reported to stimulate the expression of F. solani symptoms in common beans (de Tôledo-Souza et al., 2009). Porto et al. (2019) also reported that even though F. solani is one of the main pathogens that cause losses to melon crops, only 18.4% infections were caused by this pathogen alone and 81.6% in combination with R. solani, Macrophomina phaseolina Tassi, Monosporascus cannonballus Pollack & Uecker and Sclerotium rolfsii Sacc. Elarosi (1957) reported that the relations following the infection of potato tubers by F. solani in a site previously infected with Rhizoctonia showed synergistic mutualism. Chang et al. (1994) highlighted the effect of interaction between R. solani and another species of Fusarium (F. oxysporum) on inducing mortality (50%), reducing stem number and decreasing stem height of Alstroemeria spp. In another research, Dattnoff & Sinclair (1988) pointed out the additive interaction between these two pathogens in causing root rot of soybeans. Kommedahl & Young (1956) reported a significant decrease in the number of wheat seedlings infected by R. solani with an increase in infected seedlings by Fusarium spp. All these findings help support that F. solani and R. solani are acting synergistically to increase damping-off and root rot disease of T. articulata.