Factors influencing infection of mechanical wounds by Fusarium circinatum on Monterey pines (Pinus radiata)

Pitch canker, caused by the fungus Fusarium circinatum , is a disease affecting many pine tree species. In California, Pinus radiata (Monterey pine) is the principal pine host affected by pitch canker. This investigation into factors affecting infection frequency by F. circinatum of P. radiata examined the influence of: (i) wound size; (ii) relative humidity; (iii) time of inoculation; (iv) inoculum density; and (v) wound age. Wounded branches sustained significantly more infections when large-diameter (1·6 mm) rather than small-diameter (0·5 mm) wounds were made. Infection frequencies tended to be higher at 100% RH than at ambient humidity, although these differences were not statistically significant. Infection frequencies were significantly higher on branches inoculated after 17·00 h than on branches inoculated before noon. Infection frequencies were significantly higher for wounded branches spray-inoculated with 5 × 10⁷ rather than 1 × 10⁷ spores mL⁻¹. Infection frequencies of pruning wounds declined as wounds aged.     

Effect of Trichoderma viride pre-inoculation in pine species with different levels of susceptibility to Fusarium circinatum: physiological and hormonal responses

Pine pitch canker (PPC), caused by Fusarium circinatum, affects Pinus species worldwide. Although no effective solutions have yet been found to control it, there is a growing interest in using biological control agents (BCA) such as Trichoderma to avoid the application of chemical-based products. Using species with an increasing level of susceptibility to PPC (Pinus pinea, Pinus pinaster and Pinus radiata), this study aimed to evaluate the effect of Trichoderma viride pre-inoculation on disease development, assessing several physiological and hormonal parameters. A 2-week period elapsed between T. viride and F. circinatum inoculation. Sampling for each species was performed independently when at least 50% of the plants of one of the inoculated groups developed disease symptoms. Fusarium circinatum infection reduced water status and photosynthesis, but increased proline, abscisic acid and jasmonic acid concentrations in plants of P. radiata and P. pinaster with symptoms; while in P. pinea water relations were maintained and anthocyanin accumulation occurred in the presence of F. circinatum. In P. radiata, T. viride pre-inoculation accelerated disease progression, with some PPC-induced responses augmented (decreased water potential and photosynthesis; increased substomatal CO₂ concentration) and novel changes not found in seedlings inoculated exclusively with F. circinatum (increased electrolyte leakage and salicylic acid; decreased relative water content). This suggests that T. viride may be initially recognized as an invading organism, subverting the plant defence mechanisms for successful root colonization. If seedlings are not allowed to recover from this state, pathogen infection may thus be facilitated, highlighting the importance of application timing in BCA strategies.     

Survival of Fusarium circinatum in soil and Pinus radiata needle and branch segments

Survival of Fusarium circinatum in colonized pine needles and wood pieces was measured. Naturally colonized branches and their needles were cut into small pieces and placed in mesh bags on the soil surface at two locations in northern Spain. Pieces were recovered periodically, cultured on a selective medium, and microscopically examined to identify the species. After 507 days, F. circinatum was recovered from 0 to 27% of the wood pieces and from none of the needles. After 858 days, F. circinatum was not recovered from any wood pieces but was found to be present on 1 out of 220 needle pieces analysed. Artificially infested pieces of wood and needles were placed on 5‐mm sieved soil either in plastic boxes at controlled temperature or in mesh bags under field conditions. No survival was recorded after 794 days under field conditions and the decline over time occurred more rapidly in inoculated pieces under field conditions. Soil was also infested with conidia of F. circinatum and survival was estimated. No conidia were recovered after 224 days at 30 °C, although at 20 and 5 °C the respective populations were 20 and 3700 cfu/g soil. Fusarium circinatum was not recovered from 2‐mm‐sieved soil collected under pitch canker‐infected pines. Results indicate that branch segments and needles naturally colonized by F. circinatum will not be a potential source of inoculum, and the fungus in soil is not likely to contribute to reinfection of new plantations after 2 years.     

Effect of mycoviruses on the virulence of Fusarium circinatum and laccase activity

Laccase enzymes (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) play a major role in the degradation of phenolic compounds such as lignin. They are common in fungi and have been suggested to participate in host colonization by pathogenic fungi. Putative mycoviruses have recently been isolated from the causal agent of pine pitch canker disease, Fusarium circinatum Nirenberg & O'Donell. In this study, the effects of single and double mycoviral infections on laccase activity, growth rate and pathogenicity were investigated in fourteen F. circinatum strains. Extracellular laccase activity was analyzed by the Bavendamm test, image processing and a spectrophotometric method. Mycelial growth, in vivo pathogenicity and seedling survival probability were also determined in Monterrey pine (Pinus radiata D. Don) seedlings. The findings showed that (i) mycelial growth of isolates from the same fungal population was homogeneous, (ii) the presence of mycovirus appears to increase the virulence of fungal isolates, (iii) co-infection (with two mycoviruses) caused cryptic effects in fungal isolates, and (iv) laccases embody a possible auxiliary tool in fungal infection. The prospects for biocontrol, the adaptive role of F. circinatum mycoviruses and the importance of laccase enzymes in host colonization are discussed.     

Inheritance of virulence in Fusarium circinatum,the cause of pitch canker in pines

Wildtype strains of Fusarium circinatum, the causal agent of pitch canker, were crossed to obtain an F₁ generation. Progeny of this cross were tested for virulence by inoculating Pinus radiata seedlings, and were found to induce a wide range of lesion lengths. Two strains from the F₁ generation that induced long lesions (= high virulence) were used as parents to produce an F₂ generation, followed by a second round of selection for high virulence to obtain an F₃ generation. Mean lesion lengths were not significantly different between the three generations (P ≥ 0.196). A parallel set of crosses was performed to select for low virulence by using progeny in the F₁ and F₂ generations that induced short lesions as parents for F₂ and F₃ generations, respectively. In this case, both rounds of selection resulted in a significant reduction in mean lesion length, from 33.8 ± 0.8 mm in the F₁ generation, to 19.7 ± 0.7 and 12.9 ± 0.7 mm in the F₂ and F₃ generations, respectively. Thus it is apparent that F. circinatum retains the genetic capacity for avirulence to pines, which could reflect a lack of strong selection for virulence in nature. Progeny of a cross between high and low virulence parents manifested nearly continuous variation in lesion lengths, consistent with virulence being a quantitatively inherited trait. Based on this cross, broad‐sense heritability (H²) was determined to be 0.74, which suggests that virulence is under strong genetic control.     

Association of the pitch canker fungus, Fusarium subglutinans f.sp. pini, with Monterey pine seeds and seedlings in California

The causal agent of pitch canker disease of pines, Fusarium subglutinans f.sp. pini , is a recent introduction to California. The work presented here demonstrates that this pathogen is seed-borne in Monterey pine ( Pinus radiata ). The pathogen was isolated from up to 83% of seeds collected from cones on recently infected branches. Seedling emergence from infested Monterey pine seeds was 9%, compared with 67% for uninfested seeds. The fungus was isolated more frequently from seedlings originating from diseased branches than from symptomless branches. However, more than 50% of seeds from symptomless Monterey pine branches produced symptomless seedlings from which the pathogen was isolated. It is hypothesized that F. s . pini is carried within the seeds, where it may remain dormant until germination. The implications of these findings for the regeneration of native and landscape-planted Monterey pines, and for the transport of this non-native pathogen in pine seeds and seedlings to previously uninfested areas, are discussed.     

The complex symbiotic relationships of bark beetles with microorganisms: a potential practical approach for biological control in forestry

Bark beetles, especially Dendroctonus species, are considered to be serious pests of the coniferous forests in North America. Bark beetle forest pests undergo population eruptions, causing region wide economic losses. In order to save forests, finding new and innovative environmentally friendly approaches in wood-boring insect pest management is more important than ever. Several biological control methods have been attempted over time to limit the damage and spreading of bark beetle epidemics. The use of entomopathogenic microorganisms against bark beetle populations is an attractive alternative tool for many biological control programmes in forestry. However, the effectiveness of these biological control agents is strongly affected by environmental factors, as well as by the susceptibility of the insect host. Bark beetle susceptibility to entomopathogens varies greatly between species. According to recent literature, bark beetles are engaged in symbiotic relationships with fungi and bacteria. These types of relationship are very complex and apparently involved in bark beetle defensive mechanisms against pathogens. The latest scientific discoveries in multipartite symbiosis have unravelled unexpected opportunities in bark beetle pest management, which are discussed in this article.