Herbivores equalize the seedling height growth of three dominant tree species in an African tropical rain forest

Determining the impact of insect herbivores on forest tree seedlings and saplings is difficult without experimentation in the field. Moreover, this impact may be heterogeneous in time and space because of seasonal rainfall and canopy disturbances, or ‘gaps’, which can influence both insect abundance and plant performance. In this study we used fine netting to individually protect seedlings of Microberlinia bisulcata, Tetraberlinia bifoliolata and Tetraberlinia korupensis trees (Fabaceae = Leguminosae) from insects in 41 paired gap-understorey locations across 80 ha of primary rain forest (Korup, Cameroon). For all species, growth in height and leaf numbers was negligible in the understorey, where M. bisulcata had the lowest survival after c. 2 years. In gaps, however, all species responded positively with pronounced above-ground growth across seasons. When exposed to herbivores their seedling height growth was similar, but in the absence of herbivores, M. bisulcata significantly outgrew both Tetraberlinia species and matched their leaf numbers. This result suggests that insect herbivores might play an important role in maintaining species coexistence by mitigating sapling abundance of the more palatable M. bisulcata, which in gaps was eaten the most severely. The higher ratio in static leaf damage of control-to-caged M. bisulcata seedlings in gaps than understorey locations was consistent with the Plant Vigour Hypothesis. This result, however, did not apply to either Tetraberlinia species. For M. bisulcata and T. korupensis, but not T. bifoliolata (the most shade-tolerant species), caging improved relative seedling survival in the understory locations compared to gaps, providing restricted support for the Limiting Resource Model. Approximately 2.25 years after treatments were removed, the caged seedlings were taller and had more leaves than controls in all three species, and the effect remained strongest for M. bisulcata. We conclude that in this community the impact of leaf herbivory on seedling growth in gaps is strong for the dominant M. bisulcata, which coupled to a very low shade-tolerance contributes to limiting its regeneration. However, because gaps are common to most forests, insect herbivores may be having impacts upon functionally similar tree species that are also characterized by low sapling recruitment much more widely than currently appreciated. An implication for the restoration and management of M. bisulcata populations in forests outside of Korup is that physical protection from herbivores of new seedlings where the canopy is opened by gaps, or by harvesting, should substantially increase its subcanopy regeneration, and thus, too, its opportunities for adult recruitment.

Rhizoctonia -Scots pine interactions: detection, impact on seedling performance and host defence gene response

Rhizoctonia spp. are ubiquitous soil inhabiting fungi that enter into pathogenic or symbiotic associations with plants. In general Rhizoctonia spp. are regarded as plant pathogenic fungi and many cause root rot and other plant diseases which results in considerable economic losses both in agriculture and forestry. Many Rhizoctonia strains enter into symbiotic mycorrhizal associations with orchids and some hypovirulent strains are promising biocontrol candidates in preventing host plant infection by pathogenic Rhizoctonia strains. This work focuses on uni- and binucleate Rhizoctonia (respectively UNR and BNR) strains belonging to the teleomorphic genus Ceratobasidium, but multinucleate Rhizoctonia (MNR) belonging to teleomorphic genus Thanatephorus and ectomycorrhizal fungal species, such as Suillus bovinus, were also included in DNA probe development work. Strain specific probes were developed to target rDNA ITS (internal transcribed spacer) sequences (ITS1, 5.8S and ITS2) and applied in Southern dot blot and liquid hybridization assays. Liquid hybridization was more sensitive and the size of the hybridized PCR products could be detected simultaneously, but the advantage in Southern hybridization was that sample DNA could be used without additional PCR amplification. The impacts of four Finnish BNR Ceratorhiza sp. strains 251, 266, 268 and 269 were investigated on Scot pine (Pinus sylvestris) seedling growth, and the infection biology and infection levels were microscopically examined following tryphan blue staining of infected roots. All BNR strains enhanced early seedling growth and affected the root architecture, while the infection levels remained low. The fungal infection was restricted to the outer cortical regions of long roots and typical monilioid cells detected with strain 268. The interactions of pathogenic UNR Ceratobasidium bicorne strain 1983-111/1N, and endophytic BNR Ceratorhiza sp. strain 268 were studied in single or dual inoculated Scots pine roots. The fungal infection levels and host defence-gene activity of nine transcripts [phenylalanine ammonia lyase (pal1), silbene synthase (STS), chalcone synthase (CHS), short-root specific peroxidase (Psyp1), antimicrobial peptide gene (Sp-AMP), rapidly elicited defence-related gene (PsACRE), germin-like protein (PsGER1), CuZn- superoxide dismutase (SOD), and dehydrin-like protein (dhy-like)] were measured from differentially treated and un-treated control roots by quantitative real time PCR (qRT-PCR). The infection level of pathogenic UNR was restricted in BNR- pre-inoculated Scots pine roots, while UNR was more competitive in simultaneous dual infection. The STS transcript was highly up-regulated in all treated roots, while CHS, pal1, and Psyp1 transcripts were more moderately activated. No significant activity of Sp-AMP, PsACRE, PsGER1, SOD, or dhy-like transcripts were detected compared to control roots. The integrated experiments presented, provide tools to assist in the future detection of these fungi in the environment and to understand the host infection biology and defence, and relationships between these interacting fungi in roots and soils. This study further confirms the complexity of the Rhizoctonia group both phylogenetically and in their infection biology and plant host specificity. The knowledge obtained could be applied in integrated forestry nursery management programmes.

Automated system of seedling image analysis (SVIS) and electrical conductivity to assess sun hemp seed vigor

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Endophytic microorganisms from coffee tissues as plant growth promoters and biocontrol agents of coffee leaf rust

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of flooding on survival and initial growth of Ocotea pulchella (Nees) Mez. seedlings in semi-controlled light conditions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Growth of euterpe oleracea seedlings under different color nets and light conditions

Euterpe oleracea, a native palm to the Amazon, has recognized trading importance because of its fruits and heart of palm. However, it still requires general information on its propagation and cultivation. As light is essential for plant growth, this work aimed to study the effect of different color nets and light conditions on the initial growth of E. oleracea, with the objective to obtain high quality seedlings. The experiment was conducted in Jaboticabal County, São Paulo State, Brazil. The experimental design was entirely randomized, with six treatments (six light conditions: red net with 50% of shading - red Chromatinet®; blue net with 50% of shading - blue Chromatinet®; black nets with 70, 50 or 30% of shading; and full sun) and six replications (six evaluation periods: 30, 60, 90, 120, 150 and 180 days after the beginning of the experiment). Each plot consisted of four pots with one plant per pot, resulting in 144 plants. Seedling growth was monthly evaluated by collecting four plants per replication until the end of the experiment. Shoot length, root length, stem diameter, leaf number, leaf area and root and shoot dry matter were evaluated. The best quality seedlings of E. oleracea were obtained when they were cultivated under the black net with 50% of shading.

Endophytic microorganisms isolated from coffee leaves, roots and branches as plant growth promoters and biocontrol agents of coffee leaf rust

Suppression of plant diseases and growth promotion due to the action of endophytic microorganisms has been demonstrated in several pathosystems. Experiments under controlled conditions involving 234 endophytic bacteria and fungi isolated from coffee leaves, roots and branches were conducted with the objective of evaluating the germination inhibition of Hemileia vastatrix urediniospores, the control of coffee leaf rust development in tests with leaf discs and on plastic bags seedling, and to promote growth of coffee seedlings. None of the fungal isolates induced plant growth or reduced disease severity. The bacterial isolates (identified by the fatty acids profile analysis) 85G (Escherichia fergusonii), 161G, 163G, 160G, 150G (Acinetobacter calcoaceticus) and 109G (Salmonella enterica) increased plant growth, the maximum being induced by 85G. This isolate produced in vitro phosphatase and indol acetic acid. In assay to control rust on coffee leaf disc, nine bacterial isolates, 64R, 137G, 3F (Brevibacillus choshinensis), 14F (Salmonella enterica), 36F (Pectobacterium carotovorum), 109G (Bacillus megaterium), 115G (Microbacterium testaceum), 116G and 119G (Cedecea davisae) significantly reduced disease severity, when applied 72 or 24h before challenging with the pathogen. In seedling tests most disease severity reduction was achieved by the isolates 109G and 119G. There was no correspondence between the organisms that promoted seedling growth and those that reduced rust severity on seedlings or leaf discs.

Automated system of seedling image analysis (SVIS) and electrical conductivity to assess sun hemp seed vigor

The development of new procedures for quickly obtaining accurate information on the physiological potential of seed lots is essential for developing quality control programs for the seed industry. In this study, the effectiveness of an automated system of seedling image analysis (Seed Vigor Imaging System - SVIS) in determining the physiological potential of sun hemp seeds and its relationship with electrical conductivity tests, were evaluated. SVIS evaluations were performed three and four days after sowing and data on the vigor index and the length and uniformity of seedling growth were collected. The electrical conductivity test was made on 50 seed replicates placed in containers with 75 mL of deionised water at 25 ºC and readings were taken after 1, 2, 4, 8 and 16 hours of imbibition. Electrical conductivity measurements at 4 or 8 hours and the use of the SVIS on 3-day old seedlings can effectively detect differences in vigor between different sun hemp seed lots.

Assessment of physiological potential of cucumber seeds using the software Seedling Vigor Imaging System® (SVIS®)

The software Seed Vigor Imaging System (SVIS®), has been successfully used to evaluate seed physiological potential by automated analyses of scanned seedlings. In this research, the efficiency of this system was compared to other tests accepted for assessing cucumber (Cucumis sativus L.) seed vigor of distinct seed lots of Supremo and Safira cultivars. Seeds were subjected to germination, traditional and saturated salt accelerated aging, seedling emergence, seedling length and SVIS analyses (determination of vigor indices and seedling growth uniformity, lengths of primary root, hypocotyl and whole seedlings). It was also determined whether the definition of seedling growth/uniformity ratios affects the sensitivity of the SVIS®. Results showed that analyses SVIS have provided consistent identification of seed lots performance, and have produced information comparable to those from recommended seed vigor tests, thus demonstrating a suitable sensitivity for a rapid and objective evaluation of physiological potential of cucumber seeds. Analyses of four-days-old cucumber seedlings using the SVIS® are more accurate and growth/uniformity does not affect the precision of results.

Seed fate and seedling dynamics after masting in two African rain forest trees

How the effects of biotic factors are moderated by abiotic factors, and their consequences for species interactions, is generally understudied in ecology. A key abiotic feature of forests is regular canopy disturbances that create temporary patches, or “gaps,” of above-average light availability. Co-occurring in lowland primary forest of Korup National Park (Cameroon), Microberlinia bisulcata and Tetraberlinia bifoliolata are locally dominant, ectomycorrhizal trees whose seeds share predator guilds in masting years. Here, we experimentally tested the impact of small mammal predators upon seedling abundance, growth, and survivorship. In 2007, we added a fixed density of seeds of each species to exclosures at 48 gap–understory locations across 82.5 ha within a large Microberlinia grove, and at 15 locations outside it. For both species, small mammals removed more seeds in gaps than in understory, whereas this was reversed for seeds killed by invertebrates. Nonetheless, Microberlinia lost twice as many seeds to small mammals, and more to invertebrates in exclosures, than Tetraberlinia, which was more prone to a pathogenic white fungus. After six weeks, both species had greater seedling establishment in gaps than understory, and in exclosures outside compared to exclosures inside the grove. In the subsequent two-year period, seedling growth and survivorship peaked in exclosures in gaps, but Microberlinia had more seedlings' stems clipped by animals than Tetraberlinia, and more than twice the percentage of leaf area damaged. Whereas Microberlinia seedling performance in gaps was inferior to Tetraberlinia inside the grove, outside it Microberlinia had reduced leaf damage, grew taller, and had many more leaves than Tetraberlinia. No evidence was found for “apparent mutualism” in the understory as seedling establishment of both species increased away from (>25 m) large stems of either species, pointing to “apparent competition” instead. In gaps, Microberlinia seedling establishment was lower near Tetraberlinia than conspecific adults because of context-dependent small mammal satiation. Stage-matrix analysis suggested that protecting Microberlinia from small mammals could increase its population growth rate by 0.06. In the light of prior research we conclude that small mammals and canopy gaps play an important role in promoting species coexistence in this forest, and that their strong interaction contributes to Microberlinia's currently very poor regeneration.

Effects of conifer sawdust, hardwood sawdust, and peat on soil properties and a barefoot conifer seedling development

Organic amendments are commonly used to improve tree nursery soil conditions for increased seedling growth. However, few studies compare organic amendments effects on soil conditions, and fewer compare subsequent effects on seedling growth. The effects of three organic amendments on soil properties and seedling growth were investigated at the USDA Forest Service J.W. Toumey Nursery in Watersmeet, MI. Pine sawdust (red pine, Pinus resinosa), hardwood sawdust (maple, Acer spp. and aspen, Populus spp.), and peat were individually incorporated into a loamy sand nursery soil in August, 2006, and soil properties were sampled periodically for the next 14 months. Jack (Pinus banksiana), red, and white pine (Pinus strobus) were sown into test plots in June, 2007 and sampled for growth responses at the end of the growing season. It is hypothesized; pine sawdust and peat can be used as a satisfactory soil amendment to improve soil conditions and produce high quality seedlings, when compared to hardwood sawdust in bareroot nursery soils. This study has the potential to reduce nursery costs while broadening soil amendment options. The addition of peat and pine sawdust increased soil organic matter above control soil conditions after 14 months. However, hardwood sawdust-amended soils did not differ from control soils after same time period. High N concentrations in peat increased total soil N over the other treatments. Similarly, the addition of peat increased soil matric potential and available water over all other treatments. Seedlings grew tallest with the largest stem diameter, and had the largest biomass in both control soil and soil amended with peat, compared to either sawdust treatment. Seedlings grown in peat-amended soils had higher N concentrations than those grown in soils treated with pine sawdust, though neither was different from seedlings grown in control or hardwood sawdust-amended soils. Overall, peat is a well suited organic soil amendment for the enhancement of soil properties, but no amendments were able to increase one-year seedling growth over control soils.

Seedling resistance, tolerance and escape from herbivores: insights from co-dominant canopy tree species in a resource-poor African rain forest

* Although plants can reduce the impacts of herbivory in multiple ways, these defensive traits are often studied in isolation and an understanding of the resulting strategies is incomplete. * In the study reported here, empirical evidence was simultaneously evaluated for the three main sets of traits available to plants: (i) resistance through constitutive leaf traits, (ii) tolerance to defoliation and (iii) escape in space, for three caesalpiniaceous tree species Microberlinia bisulcata, Tetraberlinia bifoliolata and T. korupensis, which co-dominate groves within the lowland primary rain forest of Korup National Park (Cameroon). * Mesh cages were placed around individual wild seedlings to exclude insect herbivores at 41 paired canopy gap and understorey locations. After following seedling growth and survival for c. 2 years, caged and control treatments were removed, leaves harvested to determine nutrient and phenolic concentrations, leaf mass per area estimated, and seedling performance in gaps followed for a further c. 2 years to quantify tolerance to the leaf harvesting. * The more nutrient-rich leaves of the weakly shade-tolerant M. bisulcata were damaged much more in gaps than the two strongly shade-tolerant Tetraberlinia species, which had higher leaf mass per area and concentrations of total phenols. Conversely, the faster-growing M. bisulcata was better able to tolerate defoliation in terms of height growth (reflushing capacity), but not at maintaining overall leaf numbers, than the other two species. * Across gaps, insect-mediated Janzen–Connell effects were most pronounced for M. bisulcata, less so for T. korupensis, and not detectable for T. bifoliolata. The three species differed distinctly in their secondary metabolic profiles. * Taken together, the results suggested a conceptual framework linking the three sets of traits, one in which the three co-dominant species adopt different strategies towards herbivore pressure depending on their different responses to light availability. This study is one of the first in a natural forest ecosystem to examine resistance to, tolerance of, and escape from herbivory among a group of co-occurring tropical tree species.

Cucumber Cotyledon Lipoxygenase during Postgerminative Growth. Its Expression and Action on Lipid Bodies

In cucumber (Cucumis sativus), high lipoxygenase-1 (LOX-1) activity has been detected in the soluble fraction prepared from cotyledons of germinating seeds, and the involvement of this enzyme in lipid turnover has been suggested (K. Matsui, M. Irie, T. Kajiwara, A. Hatanaka [1992] Plant Sci 85: 23–32; I. Fuessner, C. Wasternack, H. Kindl, H. Kühn [1995] Proc Natl Acad Sci USA 92: 11849–11853). In this study we have investigated the expression of the gene lox-1, corresponding to the LOX-1 enzyme. LOX-1 expression is highly coordinated with that of a typical glyoxysomal enzyme, isocitrate lyase, during the postgerminative stage of cotyledon development. In contrast, although icl transcripts accumulated in tissue during in vitro senescence, no accumulation of lox-1 mRNA could be observed, suggesting that lox-1 plays a specialized role in fat mobilization. LOX-1 is also known to be a major lipid body protein. The partial peptide sequences of purified LOX-1 and lipid body LOX-1 entirely coincided with that deduced from the lox-1 cDNA sequence. The data strongly suggest that LOX-1 and lipid body LOX-1 are derived from a single gene and that LOX-1 can exist both in the cytosol and on the lipid bodies. We constructed an in vitro oxygenation system to address the mechanism of this dual localization and to investigate the action of LOX-1 on lipids in the lipid bodies. LOX-1 cannot act on the lipids in intact lipid bodies, although degradation of lipid body proteins, either during seedling growth or by treatment with trypsin, allows lipid bodies to become susceptible to LOX-1. We discuss the role of LOX-1 in fat mobilization and its mechanism of action.

Effects of nitrogen source and ectomycorrhizal association on growth and delta N-15 of two subtropical Eucalyptus species from contrasting ecosystems

Ectomycorrhizal (EM) associations facilitate plant nitrogen (N) acquisition, but the contribution of EM associations to tree N nutrition is difficult to ascertain in ecosystems. We studied the abilities of subtropical EM fungi and nutritionally contrasting Eucalyptus species, Eucalyptus grandis W. Hill ex Maiden and Eucalyptus racemosa Cav, to use N sources in axenic and soil cultures, and determined the effect of EM fungi on plant N use and plant N-15 natural abundance (delta N-15). As measured by seedling growth, both species showed little dependence on EM when growing in the N-rich minerotrophic soil from E. grandis rainforest habitat or in axenic culture with inorganic N sources. Both species were heavily dependent on EM associations when growing in the N-poor, organotrophic soil from the E. racemosa wallum habitat or in axenic culture with organic N sources. In axenic culture, EM associations enabled both species to use organic N when supplied with amide-, peptide- or protein-N. Grown axenically with glutamine- or protein-N, delta N-15 of almost all seedlings was lower than source N. The delta N-15 of all studied organisms was higher than the N source when grown on glutathione. This unexpected N-15 enrichment was perhaps due to preferential uptake of an N moiety more N-15-enriched than the bulk molecular average. Grown with ammonium-N, the delta N-15 of non-EM seedlings was mostly higher than that of source N. In contrast, the delta N-15 of EM seedlings was mostly lower than that of source N, except at the lowest ammonium concentration. Discrimination against N-15 was strongest when external ammonium concentration was high. We suggest that ammonium assimilation via EM fungi may be the cause of the often observed distinct foliar delta N-15 of EM and non-EM species, rather than use of different N sources by species with different root specialisations. In support of this notion, delta N-15 of soil and leaves in the rainforest were similar for E. grandis and co-occurring non-mycorrhizal Proteaceae. In contrast, in wallum forest, E. racemosa leaves and roots were strongly N-15-depleted relative to wallum soil and Proteaceae leaves. We conclude that foliar delta N-15 may be used in conjunction with other ecosystem information as a rapid indicator of plant dependency on EM associations for N acquisition.

Factors affecting the nodulation and growth of tropical woody legume seedlings

This study surveys the occurrence of nodulation in woody legume species in Panamá and Costa Rica, describes nodule and root characteristics, and researches host-bacteria specificity, nodulation potential of soils, and the effects of light, added nitrogen, and rhizobia and VA mycorrhizal fungi inoculation on seedling growth. I examined 83 species in 37 genera and found 80% to be nodulated. Percent nodulated species in the Caesalpinioideae, Mimosoideae, and Papilionoideae was 17, 95, and 86, respectively, with no correlation between nodule morphology and tribal classification. Nodules formed mainly at root branch points which supports epidermal breaks as an important rhizobia infection route. More non-nodulated than nodulated species had root hairs. Several species emitted volatile sulfur-containing compounds, including the toxic compound ethylmercaptan, from roots, germinating seeds, and other tissues. These emissions may have an allelopathic action against pathogens, predators, or other plants. In contrast to the general non-specificity of most legumes for rhizobia, Mimosa pigra L. was highly specific and only nodulated in flooded soils. This species' specificity, combined with a limited occurrence of its root nodule bacteria may limit its natural distribution, but its spread as an invasive weed is facilitated when fill material from rivers is deposited in other areas. ^ An experimental light level of 1.5% of full sun completely inhibited seedling nodulation, as do similar naturally low levels in forest understory. In the forest, trees and seedlings were not nodulated. in some soils with suspected high N content. For six experimental species, added N progressively increased seedling growth while decreasing nodule biomass; at the highest level of added N nodulation was completely suppressed. Species and individuals showed variation in nodule biomass at high N applications which may indicate an opportunity for genetic selection for optimal N acquisition. Rhizobia inoculation had a small positive effect on seedling shoot growth, but VA mycorrhiza inoculation overwhelmingly increased seedling size, biomass, and leaf mineral concentration. In lowland tropical forest, VA mycorrhizal colonization appears indispensable for legume nodulation because of the fungus' ability to supply P in deficient soils. This requirement makes the legume-rhizobia-mycorrhiza association obligately tripartite. ^