Growth and photosynthetic performance of Fusarium solani infected Cucumis sativus L. treated with Trichoderma asperellum

Fusarium wilt disease caused by Fusarium species is a serious soilborne fungal disease that threatens the production of cucurbits in Malaysia. Despite multiple controls and measures, this economically important pathogenic genus continues to damage crops. Therefore, this study aims to compare the photosynthetic performance and to evaluate the gas exchange measurement of Fusarium solani-infected cucumber grown in the presence of Trichoderma asperellum, a biocontrol agent of Fusarium. Soil was infested with T. asperellum B1902 before planting cucumber seed inoculated or not inoculated with Fusarium. Thirty days post-inoculation (dpi), plant grown with T. asperellum B1902 had longer stems, more leaves and greater leaf area than that of the infected control. Furthermore, plants grown with T. asperellum B1902 had more efficient photosystem II (PSII) and enhanced photosynthetic performance. The data collected from this study contribute to our understanding of some of the mechanisms at play in the cucumber-Trichoderma–Fusarium interaction.


Introduction
The biological control of Fusarium wilt disease of plants including cucumber, banana and tomato has become an attractive alternative to chemical fungicides and other conventional control tactics [1].The genus Trichoderma has gained a lot of attention due to its biological efficacy against multiple deadly plant pathogens, including pathogenic Fusarium species [2].Several mechanisms have been reported to explain the ability of this genus to control plant pathogen infections.These include the production of cell wall degrading enzymes, induction of the plant defence system and production of antibiotics [3][4][5].Much of the research to date has emphasized molecular and defence signalling inductions.There is limited focus on physiological parameters of Fusarium wilt-infected cucurbits with Trichoderma species.In Malaysia, some experiments have been carried out for the biological management of soil-borne phytopathogens with native Trichoderma strains [6][7].
Inoculation of Trichoderma either in axenic systems or directly to the soils affects the root system architecture.Trichoderma enhanced the root biomass and increased root hair development.Roots play a major role in plant fitness through anchorage, water use efficiency and acquisition of mineral nutrients from the soil [8].Chlorophyll fluorescence, leaf conductance and hormone regulations can be monitored with current technology [9][10][11].We hypothesize that Trichoderma enhances photosynthesis and affects the growth of cucurbits.Hence, this study aims to uncover the physiological changes caused by Trichoderma species in Fusarium-infected cucurbits.

Fungal strains and inoculum preparation
Trichoderma asperellum B1902 was obtained from the Laboratory of Mycology, Department of Biology, Faculty of Science, Universiti Putra Malaysia (UPM).This strain, B1902, was originally isolated from the soil of banana plants in Dengkil, Selangor.This isolate showed the most potential [12] to control banana wilt caused by F. oxysporum f. sp.cubense, isolate 9888 [12].Fusarium solani M1799C was isolated from Fusarium wilt-infected cucumber in Bukit Rambai, Melaka, Malaysia.The isolate was identified at species level based on morphological, beta-tubulin (β-TUB) and translation elongation factor (TEF) 1α sequence analysis.The β-TUB and TEF-1α gene sequences of F. solani M1799C were deposited to GenBank with accession nos.MK522426 and KT211617, respectively.Pathogenicity test was conducted to fulfil Koch's Postulate.
Both fungi were cultured onto potato dextrose agar (PDA) and incubated at 28 ± 2°C with fluorescent light/dark regime for 10 days (T.asperellum B1902) and 7 days (Fusarium solani M1799C) [13].Sterile distilled water (10 mL) was poured onto the matured culture and the mycelia were scratched to dislodge conidia using pipette tips.The spore suspension was diluted to a final concentration of 2.0 × 10 6 conidia/ mL using haemocytometer.

Plant materials and fungal inoculation
Plants were grown in polybags containing a total of 1 kg mixed soils.A combination of (top soil: river sand: manure = 3:2:1) was autoclaved at 121°C for 15 min [14].The sterile soils allow to cool overnight and poured into 8 cm × 10 cm polybags before seed was sown.Potting soil was infested with T. asperellum B1902 inoculum before seed was sown.Briefly, 100 mL/kg of B1902 inoculum (2 × 10 6 conidia/mL) was poured and mixed into the soil.
The cucumber variety used in this study was very susceptible to F. solani M1799C.Seeds of cucumber var.Batu (Green World Genetics Sdn.Bhd., Malaysia) were surface sterilized by soaking into 10% of sodium hypochlorite (NaOCl) once, rinsed with sterile distilled water (dH 2 O) twice, and air-dried on sterile filter paper [15].The sterilized seeds were then soaked into 100 mL of F. solani M1799C inoculum overnight.Seeds were soaked into sterile distilled water as a control.The inoculated seeds were sown into polybag containing mixed soils.
Four treatments were tested as -F/-T (seeds soaked in sterile distilled water, sown into uninfested soil), +F/-T (seeds inoculated with F. solani M1799C sown into uninfested soil), -F/+T (uninoculated seeds sown into T. asperellum B1902 infested soil) and +F/+T (F.solani M1799C-inoculated seeds sown into T. asperellum B1902 infested soil).All treatments were laid at complete randomized design (CRD) under plant house located in the Biology Department, Science Faculty, UPM.The plants were grown at 12/12 h of 32 ± 1°C days and 28 ± 2°C nights with humidity of 73.6% for 30 days.Five replications were prepared for each treatment and the experiment was repeated twice.
Thirty post-inoculation (dpi) the length of the stem, number of total leaves and area of total leaves was assessed.

Gas exchange measurements
Gas exchange is another approach in determining plant physiological status.This experiment covered three vital parameters such as stomatal resistance, leaf temperature and transpiration rate.Gas exchange measurements were collected from cucumber leaves 30-dpi using Li-Cor 1600 steady-state porometer (Licor, USA).Briefly, the sensor was placed in the center of each leaf.Measurements are collected from all leaves of each treated and control plants.The cumulative value of cucumber plant treatments was scored on scales (1-4) which the highest value gets the highest score.

Chlorophyll fluorescence measurements
Chlorophyll fluorescence measurements were collected from 30 dpi using a pocket PEA chlorophyll fluorimeter (Hansatech Instrument Ltd., UK) following procedure as stated in the previous study [16].Briefly, the measurements were recorded from each leaf of treated and control plants.Measurement of initial fluorescence (F O ), maximum fluorescence (F m ), variable fluorescence (F V ), performance index (PI) and density of reaction centres per PSII antenna chlorophyll (RC/ABS) were obtained from this procedure [17].The initial fluorescence (F o ) and maximum fluorescence (F m ) were measured under modulated red light (0.5 µm m −2 s −1 ) with 1.6 s pulses of saturating light (6.8 µm m −2 s −1 PAR).The variable fluorescence (F v ) was calculated using the formula F m -F o .The cumulative value of chlorophyll fluorescence of cucumber plant treatments was scored in scales (1-4) which the highest value gets the highest score [18].

Effect of T. asperellum B1902 on Fusarium wilt of cucumber
Fusarium wilt symptoms were observed on inoculated plants.Disease parts were stunted, lower leaves were chlorotic and the roots necrotic (Figure 1).Plants inoculated with Fusarium without Trichoderma treatment had shorter stems, less leaves and less leaf area compared to plants inoculated with Fusarium and treated with Trichoderma as shown in Table 1 [19].

Photosynthetic performance of wilt-infected cucumber treated with T. asperellum based on chlorophyll fluorescence.
As shown in Figure 2(A), the chlorophyll fluorescence was greater for +F/+T plants compared to +F/-T plants (p < 0.05).Similarly, the maximum efficiency of PSII (F v /F m ) which indicates the overall efficiency of photosynthesis, in +F/+T plants was greater than +F/-T plants, and F v /F o was 2.5-fold higher than +F/-T plants (Figure 2B).Maximum efficiency was also increased in -F/+T plants.The same result as F v /F m , -F/+T plants showed higher F o value than -F/-T.Regardless of Fusarium wilt infection, Trichoderma treatment alone results in an enhanced maximum yield of PSII.+F/-T plants recorded the highest F o value compared to any other plants.+F/-T plants recorded 822.2 ± 34.1 of F o with one fold higher than +F/+T plants (Figure 2C).F v /F m measurement made over a diurnal course could lead to information on temperature and other   2D).+F/+T plants had twofold higher PI and RC/ABS measurements than +F/-T (Figure 2D) and -F/+T also higher compared to both PI and RC/ABS (Figure 2E).

Photosynthetic performance of wilt-infected cucumber treated with T. asperellum based on gas exchange measurements
At 30 dpi, the transpiration rate of +F/+T cucumber plants was twofold higher than the transpiration rate of +F/-T plants.The stomatal resistance of infected +F/-T plants was threefold higher than +F/+T plants.However, there were no significant differences in leaf temperature among treatments (Table 1).-F/-T plants recorded the lowest stomatal resistance and the highest transpiration rate compared to all other treatments.A total of 11 parameter results in the highest score as cucumber plants treated with T. asperellum B1902 (-F/+T) with a cumulative score of 35, followed by -F/-T with a cumulative score of 34 and +F/+T with cumulative score of 30.Cucumber plants inoculated with F. solani M1799C (+F/−T) results in the lowest score of only 11 (Table 1).From the cumulative score, the analysis shows that T. asperellum B1902 treatment has high efficacy in enhancing plant growth.-F/+T plants show the highest score (4) in six out of eleven parameters which include area of total leaves, maximum efficiency of PSII (F v /F m ), maximum yield of PSII (F v /F o ), minimal fluorescence (F o ), performance index (PI) and density of reaction centre per PSII antenna chlorophyll (RC/ABS).+F/-T plants show the lowest score (1) in all parameters.This result shows that +F/-T cucumber plants were deteriorated in growth due to F. solani M1799C invasion.+F/+T cucumber plants recorded the highest score (4) in the length of stem and leaf temperature.The T. asperellum B1902 treatment on +F/+T cucumber plants increase the plant growth which resulted in a high cumulative score compared to +F/-T plants.

Discussion
The effectiveness of Trichoderma isolates can play a vital role in sustainable agriculture such as controlling phytopathogenic fungi, increasing plant growth and resistance against diseases.Findings from this study showed that T. asperellum enhanced the growth of stems, number and area of leaves, stomatal resistance and transpiration rate of treated plants.Hence, future study should be focusing more on applying T. asperellum as a biocontrol agent in the field and controlling plant diseases in agricultural plantation.
Hypothetically, plant treated with T. asperellum B1902 showed greater growth corresponding to positive control.This phenomenon could be explained by the endophytic associations and ability of Trichoderma spp. to interact with other rhizosphere microbes to influence disease protection and plant growth.Through the solubilization of nutrients and chelation of minerals available, Trichoderma increases and advances nutrient uptake efficiency [20][21].Hermosa et al. [22] discussed a profound explanation of this mechanism.Furthermore, the ability of this fungus to colonize root intercellular space also does explain this bio-control activity.Before this, it has been observed that plantderived sucrose-like cucumber is a vital source for Trichoderma cells to colonize the roots and coordinate defence mechanism [23].The cells are able to recognize and adhere to the root surface, and penetrate and withstand toxic metabolites produced by the plant in response to Trichoderma invasion.This ability is mediated by the hydrophobins, small hydrophobic proteins coated the outermost cell wall layer of the fungal cell [24].
Agriculture practice has long been manifesting the use of Trichoderma species in the soil to increase crop yield and control soil-borne pathogens worldwide.They are presently marketed as biopesticides, biofertilizers and growth enhancers [25].The researchers found the species possess innate resistance to multiple chemicals applied in agriculture such as fungicide.Recently, the involvement of metabolic components, volatile organic compounds and pathogenesis-related proteins increase the mechanical strength in multilayer protection of cucumber was activated by Trichoderma [26].
In this study, the in vivo experiment was conducted in the presence of full sunlight which is the main source of energy that steers the photosynthetic processes.In measuring the yield of chlorophyll fluorescence, we were able to measure photosynthesis efficiency.-F/+T plants recorded the highest maximum efficiency of PSII compared to -F/-T, which suggests T. asperellum application alone enhances cucumber plant growth.In most unstressed plants, the F v /F m value is highly consistent with approximated to value of 0.83.Any type of stress and damages result in a lowering of F v /F m [16].
The principle underlying chlorophyll fluorescence analysis covers almost every aspect of the photosynthetic elements of a plant.The interpretation of fluorescence kineticity is relatively straightforward.This principle privileges the rapid screening of photosynthesis resulting in high-resolution information regarding plant's status often related to growth parameters, yield potential and disease attack using various measurements within a short period [16].Fusarium wilt attacks cucumber plants resulting in deteriorated growth but effectively control by T. asperellum treatment.This research contributes to a pilot study on a photosynthetic aspect of Trichoderma treated-cucurbitaceae crops.F v /F o indicates the maximum yield of PSII.The output of decreased dark-adapted F v /F m and increased F o indicates the incident of photoinhibition damage in response to high temperature, water stress and excess photon flux density (PFD) [9,27].F o values observed in -F/-T and -F/+T plants were low.
-F/+T plants recorded the highest performance index and density of the reaction centre in the PSII antenna.Ramezami et al. [28] reported that biotic stresses such as fungal infection could also lead to the damage of PSII and other components of the electron transport chain and subsequently a decline photosynthetic efficiency.
The relative quantum efficiency of PSII ( PSII) and F v /F m of tomato plant also demonstrated the biocontrol properties against Fusarium infection when treated with T. asperellum T34 [29].In the presence of T. asperellum, plant infected with F. oxysporum had improved PSII and F v /F m which suggest T. asperellum enhances photosynthesis.Fusarium solani a common causal agent of plants was previously reported to decline the F v /F m value of most other plants as well such as apple [30].Pathogenic Fusarium blocked electron transport, damage the cellular plasma membrane, increased reactive oxygen species (ROS) and restricted growth in cucumbers [11].
Trichoderma asperellum B1902 reduced the stomatal resistance of +F/+T plants and increased the transpiration rate.Stomatal resistance was greater in Fusariuminfected (+F/-T) plants resulting in a lower transpiration rate.Wilt symptoms in cucumber plants infected with Fusarium were ascribed to vessel plugging and systemic toxicity [31].Water is absorbed by the root and travels along the xylem vessels together with the nutrients.The cohesion-tension of water molecules through the xylem empowers the evaporation of water molecules from the leaf surfaces resulting in a pull-on adjacent water molecule creating a non-stoppable flow of water out to the atmosphere [32].
During the early stage of Fusarium infection, the transpiration rate is decreased, and leaf temperature is increased.Once the cells death occurred, water is lost accompanied by a decrease in leaf temperature.This special temperature pattern effects on disease severity are governed by the light-dark cycle of the plant.However, towards the final of infection, the infected plant's loss water balance due to dehydration of dead tissues.Thus the leaf temperature increased again.
Plant grown in the presence of Trichoderma showed enhanced growth compared to plant growth in the absence of Trichoderma.It competes and defeats other microbes like pathogenic fungus for space and nutrient leaving none available for them to grow [8].The presence of Trichoderma alone induces growth by the production of phytohormones [8,33].The plants detected changes even in the slight adaptation in the natural phenomenon with the presence of Trichoderma which result in the induction of hormone regulation.

Conclusion
The in vivo assessment of T. asperellum B1902 against F. solani M1799C infection on cucumber plants successfully characterized the biocontrol efficacy.Thirty dpi of T. asperellum B1902 infestation on the cucumber soil is sufficient time allocation of the biocontrol efficiency.Plants grown with T. asperellum had longer stems, more leaves and greater leaves area compared to infected plants, moreover, photosynthetic performance was also enhanced.Trichoderma asperellum B1902 enhanced photosynthetic performance and reduced the severity of infected plants through in vivo assessment.An infected cucumber plant with F. solani M1799C was determinately reducing growth.The plants lose their biological growth and photosynthetic efficiency.

Figure 2 .
Figure 2. Photosynthetic performance of wilt infected cucumber treated with T. asperellum B1902 based on chlorophyll fluorescence: (A) maximum efficiency of photosystem II (F v /F m ), (B) maximum yield of photosystem II (F v /F o ), (C) minimal fluorescence (F o ), (D) performance index (PI), (E) density of reaction centre per PSII antenna chlorophyll (RC/ABS).The data are shown as the mean ± SD of five replicates.The significant differences (P < 0.05) among the treatments are indicated by different letters above each bar.-F/-T (seeds soaked in sterile distilled water, sown into uninfested soil), +F/-T (seeds inoculated with F. solani M1799C sown into uninfested soil), -F/+T (uninoculated seeds sown into T. asperellum B1902 infested soil) and +F/+T (F.solani M1799C-inoculated seeds sown into T. asperellum B1902 infested soil).

Table 1 .
Stem length, number of leaves, area of leaves, gas exchange measurements and a cumulative score of cucumber plant inoculated with Fusarium, Trichoderma or both.< 0.05) among the treatments are indicated by different superscripts when compared among the same parameter.-F/-T (seeds soaked in sterile distilled water, sown into uninfested soil), +F/-T (seeds inoculated with F. solani M1799C sown into uninfested soil), -F/+T (uninoculated seeds sown into T. asperellum B1902 infested soil) and +F/+T (F.solani M1799C-inoculated seeds sown into T. asperellum B1902 infested soil).SD is a standard deviation.Values in bracket are scores (highest value gets the highest score).
1.35 ± 0.19 and 0.37 ± 0.15 respectively, compared to any other treatments and control (Figure