Effect of Trichoderma harzianum on tomato plant growth and its antagonistic activity against Phythium ultimum and Phytopthora capsici
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Effect of Trichoderma harzianum was investigated on plant growth and inhibitory activity against Phythium ultimum and Phytopthora capsici under laboratory and greenhouse conditions. Data under lab conditions revealed that mycelial growth of both pathogens were inhibited by T. harzianum in vitro. The effect of T. harzianum on different plant growth parameters was assessed in the presence of P. ultimum and P. capsici. Fresh and dry shoot weight was reduced by both fungal strains. The fresh shoot weight was decreased by 38.8 and 44.4% in case of P. capsici and P. ultimum, respectively. T. harzianum improved the overall plant growth in the presence of P. ultimum and P. capsici. Histopathological observation of P. ultimum and P. capsici infected tissue of the root clearly indicated that both severely affected the epidermis and vascular bundle of the host plant. T. harzianum reduced the size of lesions caused by the two pathogens. Observation of hyphae interaction of the T. harzianum with pathogens demonstrated that it inhibited the entry of both pathogens to the vascular bundle of the host tissue. Furthermore, no effect was observed on the vascular bundle, pith and cortex of treated host plant inoculated with T. harzianum and pathogens.
KeywordsHistopathology Trichoderma harzianum Phythium ultimum Phytopthora capsici Bio control Tomato
Tomato (Lycopersicum esculantum Mill) is an important solanaceous vegetable crop all over the world including Pakistan. In Pakistan, its productivity and yield is low as compared to other developed countries due to many reasons (Heuvelink et al. 2003). Among one of them is soil-borne fungal diseases. Phythium ultimum and Phytopthora capsici are the most common destructive soil-borne pathogens having broad host range and causes severe crop loss to the farmers (Hausbeck and Lamour 2004).
P. capsici infects the underground part of plant, causing seed rot and seed blight as well as stem blight and overall stunted plant growth. Due to its severe infection, the fruits of the plant prematurely fall with low market value. P. ultimum usually destroys the conductive tissue of the root system which impairs translocation of water and mineral to the upper part of the plant producing stunted plant growth (Hendrix and Campbell 1973). Diseases caused by these soil-borne pathogens are complex in nature; therefore, their control and management is challenging task. Use of chemical pesticides is common and rapid mean of controlling soil-borne pathogens. However, the use of these chemicals are associated with negative impact like hazards to human, damage the beneficial soil micro-organisms, development of resistance by pathogen and also cause environmental pollution (Ragunathan and Divakar 1996).
Trichoderma species have long been identified and characterized as potential opportunistic, avirulent plant symbionts and biological agent against different soil-borne pathogens (Naseby et al. 2000; Harman et al. 2004). The important features of Trichoderma species are as follows: they rapidly colonize in the soil, favor wide range of environment, easy to isolate and culture, grow rapidly on many substrates, effective against a wide range of plant pathogens and rarely pathogenic to plants (Brotman et al. 2012; Khatabi et al. 2012). Histopathological study of host pathogen and biocontrol agent is very important to understand and visualize the efficacy of biological control agents (BCA) at a cellular level. It provides the base for searching the control measures of different diseases.
The present study was conducted to assess the effect of Trichoderma harzianum and its inhibitory activity against P. ultimum and P. capsici on tomato plants under lab conditions. Moreover, the histopathology of host roots was also performed to explore the interactions of T. harzianum, P. ultimum, and P. capsici.
Materials and methods
Isolates of P. capsici, P. ultimum, and T. harzianum were obtained from the Department of Plant Pathology, The University of Agriculture, Peshawar, Pakistan.
In vitro assay
where I refers to percent inhibition, C is control, and T radial growth of pathogen (mm) in the presence of T. harzianum.
Green house experiment
Tomato germplasm, i.e. money maker, was obtained from Tarnab Agriculture Station, Peshawar, Pakistan. Nursery was raised in earthen pots from 3-week-old seedlings, transplanted to the 8.8inches diameter pots containing 2.5 kg of sterilized soil having sand, clay, and silt at a ratio of 2:1:1. The pure cultures of P. capsici, P. ultimum, and T. harzianum were refreshed on PDA media for 4 days at 25 °C. The inoculum was prepared in potato dextrose broth and placed in a shaking incubator for 1 week at 25 °C ± 2 °C. The flask containing the culture media was then seeded with disks (7 mm diameter) of 4-day-old culture (Margaret et al. 2011). Holes were made in rhizosphere, and 5 ml of conidial suspension of 107 ml− 1 was poured in each hole.
Both pathogens and biological agent were applied to the rhizosphere after 10 days of transplantation in a greenhouse of the Centre for Biotechnology and Microbiology (CB&M), University of Swat, Pakistan.
The experiment was designed in randomized complete block design (RCBD) with six treatments and three replications. The treatments were categorized in to the following:
T. harzianum (alone).
Inoculated with P. capsici (alone)
Inoculated with P. ultimum (alone)
Inoculated with P. capsici and T. harzianum
P. ultimum with T. harzianum
The experiments were terminated after 40 days of inoculation. Plants of each pot were carefully uprooted, separately labeled, and brought to the laboratory. Data on different agronomic traits were recorded and analyzed by Statistic 8.1 Program. Means of the results were compared by using least significant difference (LSD) test (Steel et al. 1997).
Roots were kept in each of the above solutions for 2 h at room temperature. Dehydrated root tissues were infiltrated and imbedded in paraffin wax at 52 °C for 10 days. Air bubbles were removed from the roots during the wax infiltration process. Sections of 12-μm thicknesses were cut with a rotary microtome which were then affixed on slides with the help of Mayer’s Albumin adhesive and stained with safranin and fast green (Sass 1958). The stained sections were mounted in Canada balsam and examined. Photographs were taken using Olympus digital camera at 4 ×, 10 ×, and 100 × magnifications.
Results and discussion
Genus Trichoderma is mostly used as biocontrol agent against different soil-borne pathogens (Ranasingh et al. 2006; Moubarak and Abdel-Monaim 2011). Trichoderma spp. as a biocontrol agent are known to compact the plant pathogenic attack and improve the plant growth and yield by enhancing the growth hormones and increment of plant beneficial microbiome (Dubey et al. 2007; Khatabi et al. 2012).
The present evaluation clearly indicated that T. harzianum had strong antagonistic activity against P. ultimum than against P. capsici. Therefore, it could be recommended to be used for management of the diseases caused by P. ultimum and P. capsici.
We are thankful to the Higher Education Commission Pakistan (HEC) for providing financial support to this research project.
MNU conceived idea and designing of experiment. WK management of article, analysis of data and critical revision. UuR conduct experiments. NU participated in experiments design and coordination. MM drafting of manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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