Biological control of fusarium wilt of pine seedlings using endophytic microorganisms and silicon.

Abstract

Globally, pitch canker fungus (Fusarium circinatum) poses a serious threat to the softwood forest industry. In South Africa, F. circinatum has characteristically been a nursery pathogen, causing a seedling wilt, and has attacked primarily Pinus patula and P. radiata seedlings and cuttings. With P. patula being the most commercially important species in southern Africa, this creates a substantial economic problem. There are no effective control methods to date. The overall study objective, therefore, was to isolate endophytic microorganisms from healthy pine seedling and seeds, and to screen these for activity against F. circinatum in pine seedlings. A second primary objective was to test soluble silicon (Si), applied as potassium silicate for its potential to control of Fusarium wilt of pine seedlings caused by F. circinatum. For the study to be carried out, standard methodologies and procedures had to be followed, which included the isolation of the pathogen and endophytes that were used in experiments reported in the subsequent chapters. A pathogenic strain of F. circinatum was isolated. One hundred and fifty isolates of bacterial and fungal endophytes were also isolated from the seeds of seven different species of pine and 110 seedlings and cuttings of various species of pine. For the successful screening of resistant cultivars, and control agents against F. circinatum, a reliable and representative artificial inoculation technique was needed. A study was undertaken to test various inoculation techniques, aiming to develop a reliable inoculation technique that would mimic the natural infection process of F. circinatum in the field, and to investigate the spore load required to initiate disease, without applying an excessive inoculum. Three inoculation techniques were tested

using six P. patula hybrids/species. These included drenching with a conidial suspension, injection of the crown with conidia, and a wounding technique as developed by FABI, University of Pretoria, which involves cutting off a few centimetres of the apical shoot and inoculating conidia onto the wounded tissue. Using a haemocytometer, the spore load was adjusted to two inoculum levels, namely 102 and 106 conidia ml-1. The concentration of conidia had a significant effect (p<0.05) on the Area Under the Disease Progress Curve (AUDPC). A concentration of 106 conidia ml-1 caused more severe F. circinatum symptoms and more severe disease. Inoculation techniques also had a significant effect on AUDPC (p<0.001). The highest infection levels were achieved when plants were wounded by cutting of the top or by injection. However, drenching was a simple and reliable inoculation technique. The interactions between concentration of conidia and inoculation techniques was not significant (p>0.05). Choice of P. patula hybrid had a significant effect (p<0.05) on the AUDPC. There was a significant interaction (p<0.05) between the hybrids and the inoculation technique, where drenching was more effective at discriminating the different levels of resistance of the six tested hybrids. Wounded seedlings were all equally diseased, which reflects the failure of these two inoculation techniques to provide satisfactory inoculation. Endophytic microorganisms that were isolated from selected healthy pine seedlings, cuttings and seeds were screened for their potential as biological control agents against Fusarium wilt. Young P. patula seedlings were drenched weekly for four weeks with 5 ml of each endophyte (106 c.f.u ml-1). A pathogenic strain of F. circinatum was then inoculated onto the plant and the plants were subjected to drought stress for a week. The primary screening of the endophytes produced up to 60% reduction of the disease. The eighteen best endophyte isolates were selected for further screening. In the secondary screening, Isolates E56, E8 and E51 were the most effective biological control agents, while Isolate E85 was the least effective. Isolates E 141, E12, E13 and E27 provided a limited but significant level of control.