LU Hai-ju, CHEN Dan, XIE Xin-yue, WANG Yan, ZHANG Rong-rong

Department of Life Science and Technology, Honghe College, Mengzi 661199, PRC

Abstract In order to explore whether the endophytic Trichoderma strain P3.9 of loquat has an adverse effect on indigenous fungi in loquat rhizosphere soil, the quantitative change of aboriginal fungi is determined by dish dilution and plate colony-counting method with time changing in one season. The results showed that after the inoculation of endophytic Trichoderma strain P3.9, the total number of indigenous fungi in loquat rhizosphere soil had an obviously downward trend in contrast with the control which was without inoculation. For treatment groups, 5～60 d after inoculation, the number of indigenous fungi showed a cyclic upward-downward trend at a 10-d interval except for the insignificant changes from 35 to 40 d； 60～90 d after inoculation, the upward-downward trend repeated at a 30-d interval. For the control group, the number of indigenous fungi first presented a downward-upward trend every 15 d during the period of 5～50 d, and then an upward-downward trend from the period of 50～70 d and the 75～85 d, and lastly continuous growth from 85 to 90 d； particularly, it did not vary greatly from 70 to 75 d. In general, the quantity of indigenous fungi is unstable in the control group which fluctuates more significantly than in the treatment group. The number of indigenous fungi in the treatment group was significantly lower than that in the control group. The Trichoderma strain P3.9 can inhibit indigenous fungi in loquat rhizosphere soil effectively.

Key words Loquat； Endophytic Trichoderma P3.9 strain； Indigenous fungus； Root rot

1. Introduction

Loquat (Eriobotrya japonica) is a specialty fruit in Yunnan； its flowers, fruits, leaves, roots, and barks are proved to have medical values. In 2018, the planting area of loquat in Yunnan has reached 8 500 hm2, providing an annual output value of several billions yuan (statistics from the Planting Division of Yunnan Agriculture Department) and playing an important role in the alleviation of poverty in Yunnan mountainous area. Nevertheless, the loquats have been suffering from the outbreak of root rot since 2005, and the incidence rate was up to 40%. The total number of loquats struck by root rot and the consequent economic losses are rising in recent years, resulting in a catastrophic fall in the development of loquat industry in Yunnan. The local plant protection department tried to collaborate with Honghe College to explore the method for treating loquat root rot, but no significant improvements have been made.

Symptoms of loquat root rot include the gummosis and darkening of basal cortex of the stem, the rot of basal part, scaly phloem, wilting plant, the shedding of bark, and eventually the dying of the whole plant； the onset of root rot normally reaches the peak during July to August. The duration of the disease from its onset to the death of the plant ranges from 1 month to 2 years. In earlier research, we managed to determine the pathogenic bacterium of loquat root rot. The results showed that the root rot of loquat in Yunnan Mengzi was caused byPestalotiopsis microspora[1], a destructive soil-borne pathogenic fungus that can cause severe continuous cropping obstacles. This was the first report around the world. Seedlings filled into the gap to replace the dead adult plant are impossible to survive.

YANG R Pet al.[2]conducted a traditional analysis on the microflora distribution of fungi in the rhizosphere soil of loquat plants infected by root rot. Totally, 11 genera were separated, andPenicilliumwas determined to be a dominant genus. In this paper, the endophytic fungi of loquat plant are also identified by traditional method and divided into 16 genera, among whichAcremonium,Alternaria, andPhomopsisare determined as dominant genera[3]. Specifically, aTrichodermastrain is isolated from endophytic fungi and named as P3.9； the findings proved that the bacteriostasis rate of this endophyticTrichodermastrain (P3.9) reached up to 88%[4-5]for loquat root rot fungi and 60%[6]for potted plants. P3.9 could also stimulate the growth of loquat plants, degrade cellulose and resist some of the chemical pesticides[6]. The pharmaceutical value and manorial effect of P3.9 offer great potential for practical applications. To exclude the possible adverse effects of endophyticTrichodermastrain P3.9 on loquat rhizosphere soil fungi, we inoculated P3.9 into the rhizosphere soil of loquat and calculated the number of indigenous fungi from 5 to 90 d of inoculation.

2. Materials and Methods

2.1. Materials

EndophyticTrichodermastrain P3.9, the bacterial strain for the test, was separated from the phloem of loquat trunk and preserved in the Plant Pathology Herbarium, Department of Life Science and Technology, Honghe College.

Culture medium were PDA medium (potato 200 g, glucose 16 g, agar powder 20 g, and distilled water 1 000 mL), martin medium (monopotassium phosphate 1.0 g, magnesium sulfate heptahydrate 0.5 g, peptone 5 g, glucose 10 g, agar powder 20 g, distilled water 1 000 mL, and natural pH； the mixture was heated until it boils and add 3.3 mL of 1% rose bengal solution； 0.3 mL of 1% streptomycin liquid was added into every 1 000 mL of the medium before usage). The prepared culture mediums should be sterilized at 121℃ for 30 min under high pressure. Among all the above materials, the potato was purchased from the market while other reagents were analytically pure.

2.2. Methods

2.2.1. Inoculation ofTrichodermasuspension into rhizosphere soil

Preserve the P3.9 strains on test-tube slant； transfer the strains onto a PDA plate for activation. When the dish is overgrown with the strains, use a puncher to take a 5 mm fungus cake and inoculate it onto the center of a PDA plate； cultivate the plate for 5～7 d at 28℃； make 5 culture dishes in total. When the dishes are overgrown with strains, collect all the cultures and put them into a grinder； add proper amount of distilled water； beat the mixture evenly to make fungal suspension； adjust the spore concentration to 1×106CFU/mL. Plant the grafting seedlings in nutrient bags (23×18 cm)； start the inoculation test when the seedlings are 90 d old. Irrigate the loquat root with the aboveTrichodermaspore suspension at 500 mL/plant； use same amount of clear water as the control； 10 replications； conventional irrigation and fertilization management.

2.2.2. Preparation of loquat rhizosphere soil suspension

Use a stainless cutting-ring earth auger (height 0.5 m； drill diameter 38 mm) to sample the rhizosphere soil every 5 d since the irrigation ofTrichodermaspore suspension until the 90thday； randomly select 5 plants for each group； use the method of “5-point sampling” to take rhizosphere soil (depth 5～20 cm) samples； mix the samples evenly by quartering. Weigh 1 g treatment sample and 1 g control sample； put the sample into a triangular flask containing 99 mL distilled water； shake the flasks on an oscillator for 20 min at 200 r/min； make the samples into 10-1suspension； let the suspension stand for 2 min. Draw 1 mL suspension by a sterile micropipettor and add it into 9 mL distilled water to make 10-2diluent； repeat this process to prepare 10-3～10-5diluent.

2.2.3. Counting of indigenous fungi in loquat rhizosphere soil

Take 50 μL of 10-5diluted soil suspension and add it into the martin medium by tilt-pouring； cultivate the medium at 28℃ in a thermostatic incubator； 3～5 d later, count the single colonies on the plate and calculate the number of fungi. Record the quantity changes of indigenous fungi in the treatment and control group from the 5thto the 90thday of the inoculation. The quantity of fungi can be calculated by: Fungi quantity (CFU/g of dry earth)=Mean number of colonies on each plate×dilution multiple×20× Moisture coefficient.

2.2.4. Statistics

The obtained data were processed by Excel； mean values were calculated and plotted on the chart； significance analysis were conducted for all data using SPSS19.0 and Duncan's multiple comparisons.

3. Results and Analysis

As shown in Table 1, 5～60 d after the inoculation of endophyticTrichodermastrain P3.9, the quantity of indigenous fungi in loquat rhizosphere soil of the treatment group was much lower than that of the control； generally, there was a downward trend in the quantity of indigenous fungi for the treatment group and an upward trend in that of the control. Specifically, the quantity of indigenous fungi in the treatment group showed a cyclical upward-downward trend every 10 d: an upward trend during the period of “5～10 d, 15～20 d, 25～30 d, 40～45 d, 50～55 d”, and a downward trend during the period of “10～15 d, 20～25 d, 35～40 d, 45～50 d, and 55～60 d”. For the control group, the quantity of indigenous fungi decreased during the period of “5～10 d, 20～25 d, and 35～40 d”, and increased during the period of “10～20 d, 25～30 d, and 40～60 d”.

From the 65thto 90thday of inoculation, the quantity of indigenous fungi in the treatment group was still significantly lower than that of the control. For the treatment, there was first an upward trend and then a downward trend in the number of indigenous fungi, which was quite the opposite of the control. Specifically, the quantity of indigenous fungi in the treatment group showed an upward trend during theperiod of 65～75 d and then a downward trend during 80～90 d. On the contrary, the quantity of indigenous fungi in the control decreased during the period of 65～70 d, presented no significant variations during 70～75 d, decreased during 80～85 d, and lastly increased during 85～90 d.

Table 1 Variations in fungi quantity 5～90 d after the inoculation of endophytic Trichoderma strain P3.9 into loquat rhizosphere soil ×105 CFU/g

In general, there is a downward trend in the quantity of indigenous fungi after the inoculation of endophyticTrichodermastrain P3.9 into loquat rhizosphere soil. During the period of 5～60 d after inoculation, the quantity of indigenous fungi in the treatment group showed a cyclical upward-downward trend every 10 d except for the insignificant variations during 35～40 d； during 60～90 d after inoculation, the number presented a cyclical upward-downward trend every 30 d. In the control group, the quantity of indigenous fungi showed a cyclical downward-upward trend every 15 d during the period of 5～50 d after inoculation: the quantity of indigenous fungi decreased on the first 5 d and increased on the following 10 d； from the 50thto the 70thday, the quantity of indigenous fungi showed a cyclical upward-downward trend every 20 d； there were no significant variations in the quantity during the period of 70～75 d； from the 75thto the 85thday, the quantity presented a cyclical upwarddownward trend every 10 d； there was a continuous rise in the quantity of indigenous fungi during the 85～90 d after inoculation. The fluctuation in fungi quantity of the control group was larger than that of the treatment group. It meant fungi quantity in the control was not stable. The quantity of fungi in the treatment group was much smaller that of the control, indicating effective inhibitory effects ofTrichodermastrain P3.9 on indigenous fungi.

4. Discussion

Rhizosphere microbes are not only an indispensable part of rhizosphere soils but also a double-edged sword: sometimes they are beneficial to plants, however, sometimes they could be harmful to plants' growth. Bacteria, fungi, and actinomyces are the major components of rhizosphere indigenous microbes. The quantity of bacteria ranks first in rhizosphere soil, followed by actinomyces and fungi, whereas the largest biomass is owned by indigenous fungi. There is no big relevance between the quantity of rhizosphere fungi and soil nutrients, whereas the quantity of rhizosphere fungi is relevant to soilborne diseases. The occurrence of soil-borne disease makes rhizosphere microbes tend towards “fungal type”[7]. Loquat root rot is a fungal soil-borne disease spreading through soil. The occurrence of loquat root rot is connected with the quantity of fungi in the soil[2]. Earlier research has proved that introducing endophyticTrichodermastrain P3.9 into loquat rhizosphere soils could effectively control loquat root rot[6]. In this paper, the findings indicated that endophyticTrichodermastrain P3.9 could inhibit indigenous fungi in loquat rhizosphere soils, which was conducive to loquat in its competition with root rot germs for space and nutrients[8]. These results were in accord with the existing research into the inhibitory effects ofTrichodermaon the rhizospheric indigenous fungi of lawns[9], cucumbers[10], tomatoes[11], and alfalfas[12].

As mentioned above, the microflora tends towards “fungal type”, and this stands for the increases of fungi quantity and decreases in bacteria and actinomyces, which is the major cause of soil-borne diseases[13-15]. EndophyticTrichodermastrain P3.9 could reduce the number of indigenous fungi in loquat rhizosphere soils, thus improving the quantitative dominance of bacteria and actinomyces. Consequently, the microflora in the soil would be changed from “fungal type” to “bacterial type” to boost soil material transformation[7]. These results indicated that endophyticTrichodermastrain P3.9, to a certain extent, could prevent and control loquat root rot, which were in accord with earlier research[6]. It was also reported that although the addition ofTrichodermafungicide into soils would reduce the quantity of indigenous fungi, it still increased microbial diversity[16]. And the improvement of microbial diversity was an effective method for controlling soil-borne diseases[17]. Therefore, it is also important to evaluate the influence ofTrichodermastrain P3.9 on the microbial diversity of loquat rhizosphere soils.