Histopathology of loblolly pine needles infected by Mycosphaerella dearnessii,the brown-spot needle disease pathogen

The histopathology of green Pinus taeda needles infected with Mycosphaerella dearnessii was investigated. Cellular abnormalities observed were predominately the collapse of mesophyll cells within the limits of the symptom lesions. The epidermis, hypodermis, endodermis, transfusion tissue, and the vascular system within the lesions were not affected. Resin ducts exhibited cell necrosis and dissolution within and distal from the visible limits of lesions. An intercellular interface was present in non-collapsed mesophyll cells at lesion limits, and the lateral extension of the interface usually corresponded to the effects observed in resin ducts distal from the lesions. Non-stromatic hyphae were rare in and distal from lesion tissue, indicating the host tissues were affected in advance of the pathogen. The production of a toxin by M. dearnessii is suspected.     

Histopathology of longleaf pine needles infected by Ploioderma hedgcockii (Dearn.) Darker

The histopathology of Ploioderma hedgcockii on longleaf pine needles was examined. Abnormal host tissue was confined to the visible lesion and to tissues external to the endodermis where collapse of mesophyll cells was the predominant abnormality. Resin ducts exhibited necrosis and dissolution of epithelial cells. l Hyphae, intra- and intercellular, were frequent to profuse in inter-cellular spaces resulting from mesophyll cell collapse. Hyphae were external to the endodermis and appeared limited to the symptomatic tissue by an intercellular matrix at the lesion bound-aries Hysterotheca were subepidermal and appeared seated on the hypodermis. Asci appeared contain foui “two-stage” ascospores. The presence of a toxin was not indicated in the host/pathogen relationship.     

The biology of Lophodermella conjuncta Darker on Corsican pine needles

The biology of Lophodermella conjuncta on Corsican pine needles was studied. Results of a spore exclusion experiment and histological examination of needles indicated that infection of young current years needles occurred and that the fungus first colonized only the endodermis. Infection remained latent until symptoms developed, from hyphae penetrating the mesophyll from the endodermis, in July of the year following infection. Ascocarps were produced on second year needles by mid-August. Aseospores were trapped throughout the year.Infected needles were cast prematurely during the third season. The phenomenon of latent infection of pine needles by some pine needle fungi is discussed.     

Histology of current shoots of Japanese cedar (Cryptomeria japonica D. don) inoculated withCercospora sequoiae Ellis et Everhart

The histological reaction of current shoots of the Japanese cedar,Cryptomeria japonica, inoculated withCercospora sequoiae was investigated. Wound periderm was formed on current shoots of the cedar on and after the 4th day after inoculation. In the cells of the cork layer, lignin and suberin were deposited, and a phenolic substance was accumulated. In other inoculated samples, plasmolysis, contraction of the cells and necrosis of the tissues were also observed. On the 32nd day, wound perioderm and phellogen were observed in samples of the tissue inside the necrotic tissues in response to hyphal invasion. Occurrence of wound periderm and phellogen in tissues of the needles was confirmed in the inoculated samples although no hyphae were observed in and around the tissues. These results suggest that this fungus causes necrosis of host tissues and induces defense responses. A part of this paper was presented at the 106th Annual Meeting of the Japanese Forestry Society (1995).     

First molecular detection of Lecanosticta acicola from Poland on Pinus mugo

The presence of quarantine pathogen Lecanosticta acicola, the causal agent of brown spot needle blight, was confirmed using molecular methods for the first time in northern part of Poland on the coastal area adjacent to the Baltic Sea. This area includes sandy beaches, where Pinus mugo and P. sylvestris were planted. Symptomatic needles were collected in 2017 from 20 P. mugo trees growing in one stand in Ustka. Typical symptoms of brown spot needle blight infection, including dead needle tips and central zones with yellow or reddish brown, circular spots in green tissue, were observed on all samples. Only, the asexual stage of L. acicola was obtained during this work. The pathogen species identity was confirmed using classical morphological methods (microscopic examination of the infected needles), real‐time and species‐specific priming (SSPP) PCR, and ITS sequencing. Analysis of mating‐type (MAT) genes showed the presence of both mating types in northern Poland.     

Disease biology and histopathology of bronze leaf disease: a threat to cultivated aspens,white poplars and their hybrids

Bronze leaf disease (BLD) of species and hybrids of aspens, grey and white poplars (Genus Populus, section Populus) is a systemic disease incited by the fungus Apioplagiostoma populi (E.K. Cash & A.M. Waterman) M.E. Barr that results in a characteristic bronze to dark brown pigmentation of infected leaves in late summer and early fall. Branches on affected trees die over a period of several years, disfiguring trees and eventually leading to mortality of susceptible trees planted in landscapes. In this study, we describe the histopathology of the disease and fungus development relative to external symptoms on affected trees. A. populi hyphae were observed in leaf vessels, lateral veins and xylem vessels of stems and roots of affected trees. Hyphae were also observed in veins and vessels of asymptomatic leaves. Xylem vessels of growth rings on dead branches were completely occluded with A. populi near the vascular cambium and together with the ray parenchyma were stained a bronze colour. Evidence of putative toxin damage to mesophyll cells, not colonized by hyphae, was observed and may contribute to the characteristic tissue staining observed. The development of the fungus was consistent with previous reports, but in this study, we report additional details including perithecial development and ascospore release from predominantly abaxial leaf surfaces. We also report for the first time the isolation and growth of the pathogen in pure culture and systemic infection of roots.     

Effects of foliar potassium concentration on morphology, ultrastructure and polyamine concentrations of Scots pine needles

We examined the effects of three foliar potassium concentrations (high, intermediate and low) on the morphology, ultrastructure and polyamine concentrations of current-year and 1- and 2-year-old needles of 30-year-old Scots pine (Pinus sylvestris L.) trees. Foliar K concentration had only a slight effect on needle morphology. The sclerenchyma cell walls were thinner, the xylem area was larger, and the resin ducts were smaller in needles with a low K concentration than in needles with a high or intermediate K concentration. In addition, the bundle sheath cells were collapsed in needles having a low K concentration. The secondary growth of phloem tissue and the mesophyll area were greater in needles with a high or intermediate K concentration than in needles with a low K concentration, possibly indicating greater production of photoassimilates in these trees. At the ultrastructural level, mesophyll cells with enlarged central vacuoles and small vacuoles containing electron-dense material were common in needles having a low K concentration. Enlargement of the central vacuole coincided with an exponential increase in putrescine concentration in needles with a low K concentration, suggesting that the central vacuole may function as a storage site for putrescine.     

Mycobiota in needles of Abies alba with and without symptoms of Herpotrichia needle browning

The ascomycete Nematostoma parasiticum (syn.: Herpotrichia parasitica) is commonly perceived as the causal agent of the so‐called Herpotrichia needle browning in silver fir (Abies alba). However, its fruitbodies are rarely present on symptomatic needles, which are also colonized by many presumably saprotrophic fungi. We compared the internal colonization of healthy and symptomatic needles on two sites in Poland. In addition, the endophytic mycobiota in needles of various age was recorded on two other sites without disease symptoms. Fungi were isolated from 95.6% of the dead needles and from 62.9% of the living needles on symptomatic trees, whereas on healthy trees, only 45.0% of the needles were colonized internally. Colonization frequency increased with needle age. From a total of 2017 isolates, 116 fungal taxa were identified. Frequency of many species was influenced by needle type. Anthostomella formosa, Gloeosporidiella sp., Hypoxylon fragiforme, Xylaria hypoxylon and X. polymorpha were the most common fungi isolated from living needles. In symptomless living needles, fungi occurred significantly more often in the basal than in the apical parts. In dead needles, the most common fungi were Alternaria alternata, Paraconiothyrium sporulosum, Fusarium sp., Mollisia cinerea, Rhizoctonia sp., Rhizosphaera oudemansii, Thysanophora penicillioides, Xylaria hypoxylon and X. polymorpha. Rhizoctonia sp. was the most frequently isolated fungus in dead needles (23.4%) but occurred rarely also in living needles (0.3–1.1%). The supposed pathogen N. parasiticum was detected only sporadically (at most in 0.6% of the needles). Our findings demonstrate the need for understanding the role of Rhizoctonia sp. in Herpotrichia needle browning disease aetiology.     

Excess nitrogen fertilization and the structure of Scots-pine needles

The effects of excess nitrogen fertilization on the structure of Scots-pine needles (Pinus sylvestris) were examined in Finnish Lapland. The trees were fertilized with calcium ammonium-nitrate, and the levels of nitrogen applied were 0, 75, 150, 250, 500 and 1000 kg N/ha, respectively. The thickness and width of the needle and thicknesses of the tissues were measured, and the conditions of various celltypes were classified at the light-microscopy level. In addition, the areas of the cell organelles were measured and the conditions of the chlomplasts and other cell compartments observed at the ultrastructural level. Larger needles and a thicker adaxial mesophyll were observed following excess N application (250–1000 kg N/ha). The natural xeromorphic structure of the conifer needles became more mesomorphic, possibly influencing the protection afforded against environmental stresses. A high level of nitrogen fertilization caused changes in the central cylinder, especially in the vascular bundle and the sclerenchyma. The needle nitrogen concentration was high in the highly fertilized trees and the boron concentration was simultaneously extremely low. As a consequence of the changes in the central cylinder, photoassimilate transport from the mesophyll to the phloem may be disturbed at high fertilization levels. The changes at the ultrastructural level, i.e. injuries to the chloroplast thylakoids and the plasma membrane, and the formation of lipid accumulations, were indicators of the adverse effects of excess nitrogen. With an increasing atmospheric-nitrogen load, lower nitrogen fertilization levels in pine forests naturally adapted to low soil N should be considered in forest management.     

Colonization and lignin decomposition of pine needle litter by Lophodermium pinastri

The colonization and lignin decomposition of pine (Pinus thunbergii and Pinus densiflora) needle litter by an endophytic fungus, Lophodermium pinastri, was examined with field observations, a field experiment and laboratory experiments. In pine needles collected from the field, needle mass per length and lignin content were lower in needle portions bearing Lophodermium fruiting bodies than in the remaining needle portions, whereas total carbohydrate content was not different between them. Total and live hyphal lengths were greater in needle portions bearing Lophodermium fruiting bodies than in the remaining needle portions. Lophodermium fruiting bodies were not formed on sterilized needles after a 6‐month incubation on the forest floor, whereas they formed on 20% of non‐sterilized needles, indicating that this fungus can only colonize live needles on the branch. In pure culture decomposition tests, mass loss of lignin was detected for several isolates of L. pinastri, but was variable among isolates and between the needles of two pine species. A comparison of the rDNA‐internal transcribed spacer sequences between L. pinastri isolates of P. thunbergii and P. densiflora indicated that the two groups were phylogenetically separated. This study is the first to show that L. pinastri has the ability to decompose lignin in dead pine needles.     

Plasticity in mesophyll volume fraction modulates light-acclimation in needle photosynthesis in two pines

Acclimation potential of needle photosynthetic capacity varies greatly among pine species, but the underlying chemical, anatomical and morphological controls are not entirely understood. We investigated the light-dependent variation in needle characteristics in individuals of Pinus patula Schlect. & Cham., which has 19-31-cm long pendulous needles, and individuals of P. radiata D. Don., which has shorter (8-17-cm-long) stiffer needles. Needle nitrogen and carbon contents, mesophyll and structural tissue volume fractions, needle dry mass per unit total area (M(A)) and its components, volume to total area ratio (V/A(T)) and needle density (D = M(A)/(V/A(T))), and maximum carboxylase activity of Rubisco (V(cmax)) and capacity of photosynthetic electron transport (J(max)) were investigated in relation to seasonal mean integrated irradiance (Q(int)). Increases in Q(int) from canopy bottom to top resulted in proportional increases in both needle thickness and width such that needle total to projected surface area ratio, characterizing the efficiency of light interception, was independent of Q(int). Increased light availability also led to larger M(A) and nitrogen content per unit area (N(A)). Light-dependent modifications in M(A) resulted from increases in both V/A(T) and D, whereas N(A) changed because of increases in both M(A) and mass-based nitrogen content (N(M)) (N(A) = N(M)M(A)). Overall, the volume fraction of mesophyll cells increased with increasing irradiance and V/A(T) as the fraction of hypodermis and epidermis decreased with increasing needle thickness. Increases in M(A) and N(A) resulted in enhanced J(max) and V(cmax) per unit area in both species, but mass-based photosynthetic capacity increased only in P. patula. In addition, J(max) and V(cmax) showed greater plasticity in response to light in P. patula. Species differences in mesophyll volume fraction explained most of the variation in mass-based needle photosynthetic capacity between species, demonstrating that differences in plastic adjustments in mass-based photosynthetic activities among these representative individuals were mainly associated with contrasting investments in mesophyll cells. Greater area-based photosynthetic plasticity in P. patula relative to P. radiata was associated with larger increases in M(A) and mesophyll volume fraction with increasing irradiance. These data collectively demonstrate that light-dependent increases in mass-based nitrogen contents and photosynthetic activities were associated with an increased mesophyll volume fraction in needles at higher irradiances. They also emphasize the importance of light-dependent anatomical modifications in determining needle photosynthetic capacity.     

Detection and quantification of changes in membrane-associated calcium in red spruce saplings exposed to acid fog

Five-year-old red spruce saplings (Picea rubens Sarg.) were exposed to either (1) acid fog consisting of a mixture of H(2)SO(4) and HNO(3) adjusted to pH 2.5, (2) distilled-water fog at pH 5.6, or (3) no fog (dry control) for 3.5 hours per day, five times a week during the 1996 and 1997 growing seasons. The effect of fog on cell membrane-associated calcium (mCa) of leaf mesophyll cells was investigated with the fluorescence probe chlortetracycline (CTC). In both years, mean mCa concentrations were significantly less in needles exposed to acid fog than in needles exposed to distilled-water fog or in untreated needles. In 1997, acid-fog treatment resulted in 25 and 12% reductions in mCa in current-year needles, and 18 and 15% reductions in 1-year-old needles, compared with untreated needles and needles exposed to distilled-water fog, respectively, indicating that acid deposition induced calcium leaching from the membranes of photosynthetic mesophyll cells. Exposure to distilled-water fog also led to reductions in mCa in young needles, suggesting that water films on needle surfaces can induce losses by diffusion between the needle interior and surface. Consistent with the chamber studies, field data obtained from red spruce trees at two sites in Maine showed that low mCa concentrations in needles were associated with exposure to acid fog.     

Seasonal patterns of colonization of Norway spruce needles by Lophodermium piceae

Summary Colonization of living Norway spruce needles by Lophodermium piceae was studied in southern Sweden. One‐month to 3‐year‐old needles were assessed for frequency of infection. Needles appearing in the growing season of 1996 were monitored and sampled at 1 to 4 week intervals over a period of 14 months. Infection frequency of the current‐year needles increased from around 20% in July to approximately 40% in August 1996. This level had then doubled to approximately 80% after another growing season in August 1997. Multiple infections were rare in the needles during the first growing season, but after the second growing season at least 70% of the needles had multiple infections. The average number of infections in 9‐, 21‐ and 33‐month‐old needles were 1.3, 6.3 and 8.4, respectively. The size of the individual mycelia was estimated to be small in all needles, none of the individuals could be isolated from more than two adjacent needle sections, about 1.5 mm long. In conclusion, the number of L. piceae infections per needle increased during ageing of needles but mycelial growth is probably postponed until needle senescence and death.     

New insights into the survival strategy of the invasive soilborne pathogen Phytophthora cinnamomi in different natural ecosystems in Western Australia

Despite its importance as one of the most notorious, globally distributed, multihost plant pathogens, knowledge on the survival strategy of Phytophthora cinnamomi in seasonally dry climates is limited. Soil and fine roots were collected from the rhizosphere of severely declining or recently dead specimens of 13 woody species at 11 dieback sites and two dieback spots and from healthy specimens of five woody species at four dieback‐free sites in native forests, woodlands and heathlands of the south‐west of Western Australia (WA). Phytophthora cinnamomi was recovered from 80.4, 78.1 and 100% of tested soil, fine root and soil–debris slurry samples at the 11 dieback sites, in some cases even after 18‐month storage under air‐dry conditions, but not from the small dieback spots and the healthy sites. Direct isolations from soil–debris slurry showed that P. cinnamomi colonies exclusively originated from fine roots and root fragments not from free propagules in the soil. Microscopic investigation of P. cinnamomi‐infected fine and small woody roots and root fragments demonstrated in 68.8, 81.3 and 93.8% of samples from nine woody species the presence of thick‐walled oospores, stromata‐like hyphal aggregations and intracellular hyphae encased by lignitubers, respectively, while thin‐walled putative chlamydospores were found in only 21.2% of samples from five woody species. These findings were confirmed by microscopic examination of fine roots from artificially inoculated young trees of 10 woody species. It is suggested that (i) the main function of chlamydospores is the survival in moderately dry conditions between consecutive rain events and (ii) selfed oospores, hyphal aggregations, and encased hyphae and vesicles in infected root tissue of both host and non‐host species are the major long‐term survival propagules of P. cinnamomi during the extremely dry summer conditions in WA.     

Ultrastructural study of the pathogenesis of Marssonina species to poplars

Studies on the pathogenesis of Marssonina brunnea, M. castagnei and M. populi to susceptible poplars showed that germtubes penetrated poplar leaves directly by enzymatic activity and indirectly through stomata. Six days following inoculation, hyphae had ramified throughout palisade and mesophyll tissues causing penetrated cells to become moribund. Acervuli were formed within seven days of inoculation and conidia were released by rupture of the cuticle and upper epidermal wall.     

The influence of soil scarification on the turn‐over rate of slash needles and nutrient release

The effect of soil scarification on decomposition of green Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.) needles and mineral nutrient release was investigated. The treatments studied were ploughing, disk trenching, mound on inverted humus and untreated control. The study was conducted on two clear‐felled sites with different ground moisture conditions, located in south Sweden. The needles decomposed and released their contents of N and Ca considerably faster on scarified areas than on untreated ones. For P, Mg and especially K, only minor differences between scarified and untreated areas were established. At the wet site, needle decomposition and nutrient mineralization were generally affected to the same extent by all three scarification methods studied. At the dry to fresh site, mounding and ploughing enhanced needle decomposition and nutrient mineralization more than disk trenching. The effect of different scarification methods on the long‐term production capacity of soils is discussed.     

The susceptibility of various spruce species to Rhizosphaera kalkhoffii and some cultural characteristics of the fungus in vitro

Temperature and pH requirements for mycelial growth and conidial germination were investigated for various Rhizosphaera kalkhoffii isolates. Conidial germination was also tested on spruce needles. Some factors of host susceptibility were determined using needle extract agar and buffer-capacity of needle sap.     

Co‐occurrence of the ascomycete Lophodermium piceae and the rust fungus Chrysomyxa abietis in Norway spruce needles

The frequency of needles and the proportion of needle segments infected by Lophodermium piceae were compared in symptomless and Chrysomyxa abietis‐infected, 1‐year‐old needles of Picea abies. In late spring, symptomless needles from both rust‐infected and healthy saplings were sampled. In addition, rust‐infected, totally chlorotic needles and needles with chlorosis along about half their length from the diseased trees were examined. In all three stands, the proportion of segments infected by L. piceae was larger in the rust‐infected half of the needle than in the symptomless half; but the difference was statistically significant in only one of the stands. The proportion of L. piceae‐infected segments among the nonrust‐infected needles was the same as that found for the uninfected half of rust‐infected needles (after correction for size differences). No differences in the proportion of L. piceae‐infected segments were found between the totally chlorotic, rust‐infected needles and the green needles of diseased or healthy trees.     

Physiological implications of seasonal variation in membrane-associated calcium in red spruce mesophyll cells

We examined the pattern of seasonal variation in total foliar calcium (Ca) pools and plasma membrane-associated Ca (mCa) in mesophyll cells of current-year and 1-year-old needles of red spruce (Picea rubens Sarg.) and the relationship between mCa and total foliar Ca on an individual plant and seasonal basis. Foliar samples were collected from seedlings and analyzed on 16 dated at 2- to 3-week intervals between June 1994 and March 1995. Concentrations of mCa in current-year needles were more seasonally dynamic and responsive to temporal environmental changes than either mCa concentrations of 1-year-old needles, which were largely stable, or total foliar Ca concentrations in both tissues. In current-year needles, mCa was barely evident in early summer, increased steadily through summer, and then increased dramatically in early fall and surpassed the concentration in 1-year-old needles. Coincident with the first severe frost, mCa concentrations in current-year needles declined significantly and subsequently maintained concentrations comparable to those of 1-year-old needles. Following an extended January thaw, which included 5 days of minimum temperatures > 5 degrees C, mCa concentrations of current-year needles temporarily, but significantly, declined. However, there was no change in mCa concentrations of 1-year-old needles or total Ca concentrations of either tissue. Total Ca concentrations were stable through midsummer in both tissues, doubled in late summer, and then were stable in both tissues throughout fall and winter. Total Ca concentrations were consistently higher in 1-year-old than in current-year needles. Correlations between concentrations of mCa and total foliar Ca were consistently low and mostly nonsignificant. Thus, the dominant, but insoluble, extracellular Ca pool reflected in commonly measured total foliar Ca concentrations is not a meaningful surrogate for the physiologically important and labile pool associated with the plasma membrane-cell wall compartment of red spruce mesophyll cells. It is likely that shifts in the critical mCa compartment would not be detected by analysis of total foliar Ca pools. Seasonal changes in mCa concentration seemed to parallel seasonal changes in membrane structure, and possibly the important role of extracellular Ca in transducing messages associated with environmental signals.     

Lophodermium pinastri and an unknown species of Teratosphaeriaceae are associated with needle cast in a Pinus radiata selection trial

Spring needle cast (SNC) in Tasmania is defined by the early casting of pine needles and appears in 6‐ to 7‐year‐old pine plantations after canopy closure. The severity of SNC can vary significantly among trees growing within the same plantation. Fungal communities were surveyed in a Pinus radiata family trial, using DNA extraction from needles, PCR with fungal‐specific primers and sequencing to detect the presence of fungal species. Samples were taken at the same time as the trees were scored for disease severity. Trees with contrasting levels of SNC disease severity have significantly different needle fungal communities, but family pedigree and different ages of needle are not clearly distinguished by their fungal communities. All common fungal pathogens previously implicated in causing SNC were identified from the study, but of these, only Lophodermium pinastri was correlated with high levels of disease. Several species of Teratosphaeriaceae were detected, and one of these was also strongly associated with needle cast.