The beneficial endophyte Trichoderma hamatum isolate DIS 219b promotes growth and delays the onset of the drought response in Theobroma cacao

Abstract

Theobroma cacao (cacao) is cultivated in tropical climates and is exposed to drought stress. The impact of the endophytic fungus Trichoderma hamatum isolate DIS 219b on cacao's response to drought was studied. Colonization by DIS 219b delayed drought-induced changes in stomatal conductance, net photosynthesis, and green fluorescence emissions. The altered expression of 19 expressed sequence tags (ESTs) (seven in leaves and 17 in roots with some overlap) by drought was detected using quantitative real-time reverse transcription PCR. Roots tended to respond earlier to drought than leaves, with the drought-induced changes in expression of seven ESTs being observed after 7 d of withholding water. Changes in gene expression in leaves were not observed until after 10 d of withholding water. DIS 219b colonization delayed the drought-altered expression of all seven ESTs responsive to drought in leaves by > or = 3 d, but had less influence on the expression pattern of the drought-responsive ESTs in roots. DIS 219b colonization had minimal direct influence on the expression of drought-responsive ESTs in 32-d-old seedlings. By contrast, DIS 219b colonization of 9-d-old seedlings altered expression of drought-responsive ESTs, sometimes in patterns opposite of that observed in response to drought. Drought induced an increase in the concentration of many amino acids in cacao leaves, while DIS 219b colonization caused a decrease in aspartic acid and glutamic acid concentrations and an increase in alanine and gamma-aminobutyric acid concentrations. With or without exposure to drought conditions, colonization by DIS 219b promoted seedling growth, the most consistent effects being an increase in root fresh weight, root dry weight, and root water content. Colonized seedlings were slower to wilt in response to drought as measured by a decrease in the leaf angle drop. The primary direct effect of DIS 219b colonization was promotion of root growth, regardless of water status, and an increase in water content which it is proposed caused a delay in many aspects of the drought response of cacao.

Fig. 1.

The effect of drought and DIS 219b colonization on cacao seedling growth. Seedlings were grown in magenta boxes in a controlled environment chamber (model M-2; EGC Corp., Chagrin Falls, OH, USA) with the following conditions: 12 h light/12 h dark photoperiod at 25 °C, 50% relative humidity, and 50 μmol m⁻² s⁻¹ PAR. The seedlings were either colonized with Trichoderma hamatum isolate DIS 219b or not colonized (Control). After emergence, seedlings were watered every other day for 32 d of growth in the magenta boxes. Before altering the watering cycles, the length of the hypocotyls for the non-colonized and colonized seedlings were measured. After 32 d of growth, watering was stopped for 13 d (drought treatment), while control seedlings continued to be watered every other day. (A) Hypocotyl length after 32 d of growth without drought treatment. Bars show means ±standard error. (B) The photograph of the seedlings was taken after water was withheld for 13 d.

Fig. 2.

Representative fluorescence responses of cacao leaves during drought treatment (0, 10, and 13 d without watering) of non-colonized (Control) and colonized (DIS 219b) cacao seedlings. Seedlings were watered every other day for 32 d of growth. After 32 d of growth, watering was withheld for 13 d. Fluorescence emissions of adaxial surfaces of cacao leaves at 530 nm (F530) were measured using six biological replications. Relative fluorescence intensity is given in the vertical colour scale in the middle.

Fig. 3.

Expression pattern for EST TcrbcS (putative Rubisco small subunit). Seedlings were watered every other day for 32 d of growth before drought treatment. The largest leaves were harvested 0, 7, 10, and 13 d after the last watering. Treatments: Control (water), non-colonized seedlings watered every other day; Control (no water), non-colonized seedlings with water withheld 13 d; 219b (water), colonized seedlings watered every 2 d; 219b (no water), colonized seedlings with water withheld 13 d. Relative mRNA levels were calculated for qPCR results with respect to ACTIN transcripts (% of ACTIN). Bars show means ±standard error (n=6).

Fig. 4.

Expression patterns for ESTs putatively involved in osmoprotectant production in (A) leaves including TcTPP, TcSOT, TcPR5, and TcNI and (B) roots including TcTPP, TcSOT, TcPR5, and TcCESA3. Seedlings were watered every other day for 32 d of growth before drought treatment. The roots and largest leaf were harvested 0, 7, 10, and 13 d after the last watering. Treatments: Control (water), non-colonized seedlings watered every other day; Control (no water), non-colonized seedlings with water withheld 13 d; 219b (water), colonized seedlings watered every 2 d; 219b (no water), colonized seedlings with water withheld 13 d. Relative mRNA levels were calculated for qPCR results with respect to ACTIN transcripts (% of ACTIN). Bars show means ±standard error (n=6).

Fig. 5.

Expression patterns in roots for ESTs putatively involved in hormone production (TcAOC, TcLOX), membrane function (TcABC-T, TcTIP), and other physiological processes (TcNR, TcSEN1). Treatments: Control (water), non-colonized seedlings watered every other day; Control (no water), non-colonized seedlings with water withheld 13 d; 219b (water), colonized seedlings watered every 2 d; 219b (no water), colonized seedlings with water withheld 13 d. Relative mRNA levels were calculated for qPCR results with respect to ACTIN transcripts (% of ACTIN). Bars show means ±standard error (n=6).

Fig. 6.

Expression patterns for ESTs putatively involved in signal transduction, transcription and post-transcriptional regulation. ESTs responding to drought included (A) TcMAPK3 and TcZFP in leaves, and (B) TcHK, TcRPK, TcMAPK3, TcMKK4, TcSTK, TcPP2C, and TcZFP in roots. Treatments: Control (water), non-colonized seedlings watered every other day; Control (no water), non-colonized seedlings with water withheld 13 d; 219b (water), colonized seedlings watered every 2 d; 219b (no water), colonized seedlings with water withheld 13 d. Relative mRNA levels were calculated for qPCR results with respect to ACTIN transcripts (% of ACTIN). Bars show means ±standard error (n=6).

Fig. 7.

Direct influence of DIS 219b colonization on gene expression in 9-d-old cacao seedlings germinated on 1.5% water agar plates. Colonized (219b) or non-colonized (Control) seedlings were grown for 9 d as described in the text. The whole seedling minus the cotyledons was harvested for qPCR analysis. A subset of drought-responsive ESTs was analysed. The relative mRNA levels of the ESTs were calculated with respect to ACTIN transcripts (% of ACTIN). Bars show means ±standard error (n=4).

Fig. 8.

The influence of DIS 219b colonization and watering cycle on the dry weight of cacao roots. For each treatment combination in the second biomass study, seedlings were grouped into 0.05 g weight class increments based on their root dry weights. The percentage of seedlings that died during the experiment is also included for each treatment combination. Bars show means±standard error.