Penetration of Picea sitchensis root bark by Armillaria mellea,Armillaria ostoyae and Heterobasidion annosum

Penetration of root bark tissues of Picea sitchensis by Armillaria ostoyae, Armillaria mellea and Heterobasidion annosum was examined in the absence of wounds, in superficial wounds (rhytidome tissues removed to expose the secondary phloem) and in wounds to the depth of the vascular cambium (deep wounding). Both species of Armillaria penetrated bark without prior wounding, but neither species formed rhizomorphs in this treatment. Armillaria ostoyae penetrated to 39 cell layers in depth by 48 days after inoculation of unwounded bark, whereas A. mellea penetrated 25 cell layers in the same time. Armillaria mellea penetrated superficial wounds significantly more rapidly than did A. ostoyae. Both species produced rhizomorphs within wounded host tissues. Inoculation of deep wounds with Armillaria resulted in a greater depth of bark necrosis with A. mellea than with A. ostoyae. In the absence of wounding, H. annosum failed to penetrate root bark tissues, but in both superficial and deep wounds hyphae penetrated beyond the ligno–suberized boundary zone (LSZ) by 12 days after inoculation. Where no inoculations were made, superficial or deep wounding led within 25 days to the restoration of a structurally continuous LSZ, and by day 48 the wound periderm (WP) was fully differentiated. In inoculated wounds, however, formation of the LSZ and WP was delayed or inhibited in most trees, particularly following inoculation with A. ostoyae or A. mellea. Suberization in the LSZ and WP remained diffuse and discontinuous 48 days after inoculation. Moreover, the presence of WP did not prevent further penetration of the tissues by the pathogens. Variations between trees in the depth of pathogen penetration were noted, possibly indicating differing susceptibilities of individual host genotypes. The possible host factors involved in resistance to penetration of root bark tissues by Armillaria and Heterobasidion are discussed.     

Host status effect of cowpea and sunflower on the populations ofMeloidogyne javanica andRotylenchulus reniformis

A highly susceptible cowpea,Vigna sinensis cv. Baladi plants were tested as trap plants for eitherMeloidogyne javanica orRotylenchulus reniformis under greenhouse conditions. The plants were gathered by cutting them above the surface of the soil or by uprooting them, 1/2, 1, 3, 6, 12, 24, 36 and 48 days after nematode inoculation. Both of the mentioned nematodes began to mature and lay eggs after the 12th day from their inoculation. Hence, it is advised to pull up cowpea plants from 3–12 days after nematode inoculation. After planting sunflower,Helianthus annus cv. Miak replacing cowpea, the nematode populations were higher, in most cases, on sunflower plants replacing cutting cowpea than those on sunflower replacing uprooted cowpea. The highest percentages of nematode reduction were 98.55 and 99.57 forM. javanica and 95.09 and 92.90% forR. reniformis on sunflower plants replacing cutting and uprooted cowpea plants after 12 days from nematode inoculation, respectively.M. javanica andR. reniformis decreased the length and weight of sunflower plants as affected by planting time and method of cowpea harvest. This method of nematode control is cheaper, easy and pollution free.     

Factors affecting Chondrostereum purpureum infection of Salix

Factors affecting Chondrostereum purpureum infection of Salix were studied in the laboratory and field. Basidiospores passively entered all xylem tissues but not cambial, phloem or bark tissues. A small number entered stems up to 70 mm from the wound surface although most were retained within tissues 30 mm from the surface. Basidiospores required free water for germination and the optimum temperature for germtube extension was 26°C. Hyphae ramified extensively throughout xylem tissues eventually plugging vessels. Cell walls and starch granules were penetrated with enzymatic assistance. The depth of C. purpureum penetration was affected by wound diameter and inoculum concentration but not stem age. Wounds of Salix were susceptible to C. purpureum throughout the year and greater depth of penetration was obtained using mycelium as inoculum than basidiospores. The depth of C. purpureum penetration decreased with duration of wound exposure prior to inoculation. A characteristic flora of saprophytic fungi and bacteria inhabited wound tissues.     

Immunological detection of decay in wood

Summary An enzyme-linked immunosorbent assay (ELISA) was used to detect and quantify Postia placenta (Fr.) M. Lars. et Lomb. in decaying wood. The fungus was detected as early as ten days after inoculation suggesting that ELISA may be effective in detecting early incipient decay. Decay-induced weight loss in P. placenta decayed spruce blocks was correlated with the absorbance values obtained in the ELISA reaction.     

Chemical control of two Phytophthora species infecting the canopy of Monterey pine (Pinus radiata)

Twelve active ingredients were screened for their ability to control foliage disease caused by Phytophthora kernoviae and Phytophthora pluvialis. Inhibition of mycelial growth and sporangial production of both pathogens were assessed in in vitro assays after exposure to three concentrations of each active ingredient. While most of these active ingredients inhibited mycelial growth in vitro, due to their widespread use, phosphite, copper oxychloride and metalaxyl‐M were selected for further study. Four rates of each active ingredient were applied to two Pinus radiata genotypes, and detached needle assays at 6 and 90 days post‐treatment were used to determine treatment efficacy and persistence. Untreated needles showed significantly larger lesions than all fungicide‐treated needles after exposure to P. pluvialis or P. kernoviae on both sampling dates. Efficacy and persistence of the three active ingredients did not increase with increase in concentration. Larger lesions were formed on more susceptible genotype after inoculation with both Phytophthora species, even when higher rates of active ingredients were applied. Phosphite, copper oxychloride and metalaxyl‐M have potential to protect commercially planted P. radiata from these two Phytophthora species.     

Cross‐infectivity of oil palm by Phytophthora spp. isolated from perennial crops in Malaysia

Bud rot disease or “Pudricion del cogollo” (PC) of oil palm is a major constraint on production in Colombia and neighbouring countries such as Brazil, Costa Rica, Ecuador, Nicaragua, Panama, Peru and Surinam. To date, there are no documented reports of Phytophthora disease of oil palm in South‐East Asia. This research, therefore, was conducted to determine the pathogenic potential of Phytophthora palmivora and Phytophthora nicotianae on oil palm using both in vitro and nursery inoculation experiments. In vitro inoculation of both P. palmivora and P. nicotianae on immature oil palm leaflets caused discoloration within 2 days of inoculation and incubation at 25 ± 1.5°C, 100% RH. Similarly, in nursery trials, lesions formed on the buds (unopened leaflets) 3 days after inoculation with P. palmivora or P. nicotianae zoospore suspensions. No lesions developed on untreated leaflets in either in vitro or nursery inoculation experiments. Phytophthora spp. were re‐isolated from leaflet lesions and confirmed as the inoculated pathogens.     

Effect of aging in adult Monochamus alternatus (Coleoptera: Cerambycidae) on the susceptibility of the beetle to Beauveria bassiana (Ascomycota: Hypocreales)

To clarify the effect of aging in adult Monochamus alternatus on the susceptibility of the beetle to Beauveria bassiana, we inoculated the fungus into adult beetles 0, 3, 7, 14, and 28 days after emergence. The number of days from B. bassiana inoculation to M. alternatus death increased with the number of days from beetle emergence to inoculation up to 14 days after emergence. This indicated that the susceptibility of adult beetles to the fungus decreased with aging. However, B. bassiana-inoculated beetles 28 days after emergence died earlier after inoculation than those inoculated with the fungus 14 days after emergence; i.e., the beetle’s susceptibility to the fungus increased again. We considered this phenomenon to be the result of senescence of adult beetles. We concluded that the susceptibility of M. alternatus to B. bassiana decreased first and then increased again with aging.     

Interaction of root-knot nematode and mycorrhizal fungi on common beanPhaseolus vulgaris L.

The interactions of root-knot nematode and VAM fungus on common bean plants (Phaseolus vulgaris L.), and the sequence of nematode infection were studied in the greenhouse. The inoculation with VAM fungus caused a significant increase in plant height and fresh weight compared with non treated plants.Meloidogyne infection unsignificantly decreased plant height and dry weight. When fungus was inoculated at 15 and 30 days prior toM. incognita infection, a significant increase in fresh weight was observed. There were no significant differences in total nitrogen (mg/plant) between plants inoculated withM. incognita alone or those inoculated with bothM. incognita and VAM fungus at the same time or 15 days after the fungus inoculation. Plants preinoculated with VAM fungus 30 days prior to nematode infection had a significant increase in total nitrogen. The inoculation with VAM fungus caused a significant increase in phosphorus content. However, it was significantly decreased in plants inoculated with nematode alone and in plants inoculated with VAM fungus andM. incognita at the same time. Gall index and final nematode population were significantly increased when nematodes were treated at the same time with fungus, although there were significant decrease in nematode final population and gall index when the plants were treated with nematodes at 15 and 30 days after mycorrhizal infection. A decrease in percentage of fungal colonization was observed when nematodes were inoculated with fungus at the same time.     

Responses in the bark of Cupressus sempervirens clones artificially inoculated with Seiridium cardinale under field conditions

By 70 days after inoculation, resistant and tolerant clones of field-grown Cupressus sempervirens restricted penetration of the bark by Seiridium cardinale, forming ligno-suberized boundary zones which included four to six layers of cells with suberized cell walls. This response contrasted with that in susceptible clones, where only two to four layers of suberized cells were formed in discontinuous bands around inoculation sites. Detection of multiple layers of thin suberized boundary zones in susceptible clones indicated repeated penetration of the wound/infection-induced barrier by S. cardinale during a single growing season. The methods described will be valuable in the development of rapid, early and accurate screening programmes for durable resistance to Seiridium canker in Cupressus species.     

Cultural characteristics and pathogenicity of Melanconium juglandinum

Cultural characteristics and pathogenicity of Melanconium juglandinum, the agent of the black pustular dieback of Juglans spp. in Europe were investigated. Melanconium juglandinum was consistently isolated from diseased twigs and branches of Persian (English) walnut trees (Juglans regia) in Central Italian Apennine valleys. In growth and conidia germination tests, the fungus was shown to be active within a broad range of temperatures (5–30°C), with the optimum being between 20 and 30°C. Monthly artificial inoculations performed over a period of 2 years with mycelial plugs on 2-year-old seedlings of Persian (English) walnut developed symptoms within 15 days with maximum canker length occurring when inoculation was carried out between June and August. Koch's postulates were fulfilled and M. juglandinum was proved to be a virulent pathogen.     

Effect of Betula pendula clone and leaf age on Melampsoridium betulinum rust infection in a field trial

Ten 5‐year‐old Betula pendula clones were studied for their rust resistance in the field. The trees were treated by inoculating 10 leaves on a shoot with Melampsoridium betulinum urediniospore suspension or spraying the control leaves with water. The birch clones differed significantly in their resistance to M. betulinum leaf rust fungus and the clones also varied in their responses to the local rust strain and the inoculated rust strains. However, natural rust infections and inoculation treatment were positively correlated. The older leaves had fewer infections than the younger ones on the tip of the shoot in the control trees, but in the inoculation treatment no significant correlation was found between the leaf ages and rust infection. The factors behind the different leaf susceptibilities are discussed.     

In vitro interactions between bacteria isolated from Sitka spruce stumps and Heterobasidion annosum

Culture medium composition affected antagonism by bacterial isolates from Sitka spruce (Picea sitchensis) stumps against Heterobasidion annosum. Fifty percent of bacterial isolates inhibited H. annosum growth on sporulation agar or yeast–dextrose–peptone agar; only 10% of isolates caused inhibition on both media. Proportions of isolates inhibiting H. annosum varied with stump age; fewer isolates from 4‐ or 6‐year‐old stumps exhibited antagonism than isolates from older or younger stumps. Fifteen isolates showing antagonism on sporulation agar were tested against H. annosum in spruce wood cubes. None of the bacterial isolates alone caused a significant weight reduction in inoculated cubes. Relative inoculation times of bacterial isolates and H. annosum had an effect on weight loss in interactions; simultaneous inoculation with isolates and H. annosum inhibited weight loss caused by H. annosum compared with bacteria‐free controls. Inoculation with bacterial isolates 10 days before H. annosum had no effect on the decay rate. In contrast, inoculation with H. annosum 10 days before bacteria increased weight loss of cubes by 200% relative to cultures lacking bacteria. The effect of a mixed bacterial inoculum on weight change in 0.2‐mm spruce wood slips co‐inoculated with H. annosum, Resinicium bicolor, Hypholoma fasciculare, Stereum sanguinolentum or Melanotus proteus differed between different fungi.     

Growth of Nursery-grown Bamboo Inoculated with Arbuscular Mycorrhizal Fungi and Plant Growth Promoting Rhizobacteria in two Tropical Soil Typeswith and without Fertilizer Application

A nursery experiment was conducted to assess the effect of bioinoculants (Glomus aggregatum, Bacillus polymixa, Azospirillum brasilense) on seedling growth promotion of bamboo (Dendrocalamus strictus (Roxb.) Nees.) in two soil types (alfisol, vertisol) with or without fertilizer application. Bamboo seedlings were grown in the presence or absence of bioinoculants either individually or in all combinations for 180 days in field soil under tropical nursery conditions. Shoot, rhizome and root length, dry masses, nutrient concentrations and arbuscular mycorrhizal (AM) colonized root lengths were determined at harvest. Under the experimental condition tested combined inoculation of AM fungi, PSB and A. brasilense resulted in maximum growth response both under fertilized and unfertilized conditions in both soil types. Fertilizer application enhanced the efficiencies of N, P and K uptake, whereas reduced their usage efficiencies. Though soil type did not affect microbial inoculation response, fertilizer application significantly affected plant response to microbial inoculation.     

A new leaf disease on Populus euphratica in south‐western Iran

During the winter of 2012, a leaf spot disease was observed on Euphrates poplar (Populus euphratica) in the forest areas of Khuzestan province, south‐western Iran, causing significant damage in the Karun's riverside forests. Symptoms consisted of necrotic dark brown, circular to oval, 5‐ to 10‐mm spots on both surfaces of the leaves. A fungus having distinct dictyospores similar to those produced by Alternaria spp. was observed. The morphological characteristics, as well as the phylogenetic analysis of the internal transcribed spacer (ITS1‐5,8S‐ITS2) region, confirmed the identity of the strains belonging to the species Alternaria alternata. Pathogenicity tests were conducted on the alive leaves of P. euphratica on the young branches, as well as on the detached leaves in Petri dishes, through inoculation with spore suspension. Target spot symptoms similar to those observed in naturally infected leaves were developed on the inoculated leaves seven to 10 days after inoculation in both the inoculation procedures. A. alternata was consistently re‐isolated from the spots. Interestingly, similar symptoms were observed 7 days after detached leaf treatment with droplets of 15‐day‐old fungal culture filtrate, suggesting the production of pathotoxic compounds by the fungus. To our knowledge, this is the first report of A. alternata causing leaf spot on Euphrates poplar in Iran.     

Detection of differential changes in lignin composition of elm xylem tissues inoculated with Ophiostoma novo‐ulmi using Fourier transform‐infrared spectroscopy

Differences in elm susceptibility against Ophiostoma novo‐ulmi have been related with differences in timing and degree of the tree defence responses. In this study, we used Fourier transform‐infrared spectroscopy, coupled with chemometric methods, to detect progressive changes in the metabolic profile of Ulmus minor and U. minor × U. pumila xylem tissues after inoculation with O. novo‐ulmi. Differences between control and inoculated trees were detected at 30 and 60 days post‐inoculation (dpi) in U. minor, and at 15, 30 and 60 dpi in U. minor × U. pumila. These differences were related with increased levels of lignin in the xylem tissues, suggesting an earlier defence response to the infection in the hybrids.     

Seasonal effect on infection and development of lesions caused by Cryphonectria parasitica in Castanea sativa

Seasonal variation in the development of chestnut blight, caused by Cryphonectria parasitica, was investigated by inoculating in situ chestnut trees and in vitro excised chestnut segments, at either monthly or 3‐monthly intervals throughout 30 months. Inoculations were made with conidia and mycelium of a virulent isolate and with mycelium of a hypovirulent isolate. Conidial inoculations of living sprouts or excised segments between May and July resulted in the greatest incidence of infection whereas inoculations in autumn and winter, in vitro as well as in situ, did not reveal any visible disease. However, from these symptomless inoculated stems, C. parasitica was isolated 3 months after inoculation. Inoculations with the mycelium of the virulent isolate always resulted in lesions, except in January 1999, and the greatest rate of lesion development occurred for inoculations made in the spring and summer. There was a significant seasonal effect on lesion development. Lesions caused by the hypovirulent isolate, smaller than those caused by the virulent isolate, followed a similar seasonal pattern. The same seasonal variations were observed for inoculations in vitro of excised segments. Relative water content (RWC) of chestnut bark significantly varied with bark sampling date. The rate of lesion development in sprouts significantly correlated with average minimum (ATn) and maximum (ATx) temperatures and the sum of rainfall during inoculation period, with the rate of lesion development measured in excised segments 10 days after inoculation (R10d) and with RWC measured on the day of inoculation. In multiple regression models, variables ATx and R10d best explained variation in lesion development.     

Screening of Metrosideros polymorpha (‘ōhi‘a) varieties for resistance to Ceratocystis lukuohia

Stands of the landscape‐dominant tree, Metrosideros polymorpha (‘ōhi‘a) on the Island of Hawai‘i, HI., USA, are dying from a phenomenon known as rapid ‘ōhi‘a death (ROD). Approximately 180,000 acres of forest have already been impacted by the disease, the majority of which is attributed to C. lukuohia, the more aggressive of the two Ceratocystis species responsible for ROD. Three isolates of C. lukuohia were compared for differences in aggressiveness. Mean disease severity and mean number of days to death did not differ among the three isolates of C. lukuohia. A single isolate was selected for use in a resistance screening of 128 plants across four varieties of M. polymorpha occurring on Hawai‘i Island: incana, glaberrima, polymorpha and newellii. Disease severity was lower in early‐successional var. incana (38.84%) and riparian var. newellii (36.11%) compared to late‐successional var. glaberrima (61.27%) and high‐elevation var. polymorpha (70.27%). Var. incana and var. newellii also had the lowest mortality (63% and 77%, respectively) while var. glaberrima (86%) and var. polymorpha (100%) had the highest mortality. Eighty‐five per cent of the plants that died did so within 49 days post‐inoculation and mean number of days to death was significantly higher for var. glaberrima (46) compared to var. polymorpha (31). The results of this preliminary study suggest that some form of resistance might be present in natural populations of at least one variety of M. polymorpha on Hawai‘i Island and should be investigated more intensively.     

Changes in soluble-phenol content of Norway-spruce (Picea abies) phloem in response to wounding and inoculation with Ophiostoma polonicum

The aim of this study was to investigate the temporal changes in Norway-spruce (Picea abies) phloem phenolics during wound-induced response. Two Norway spruces were inoculated with Ophiostoma polonicum and sterile agar; unwounded phloem was simultaneously tested as control. Both unwounded and inoculated phloem were then sampled 6, 11, 21, 30, and 60 days later. Monophenols were analysed by HPLC, and the tanning capacity of the phloem extracts was estimated from their ability to precipitate proteins. The response to inoculation was characterized by radical changes in the phloem phenolic composition. Initially, an increase in (+)-catechin concentration was observed, concomitant with a slow decrease in both glycoside concentrations and tanning capacity. After day 6, this decrease was accelerated in the event of fungus inoculation. The appearance of aglycones did not take place during the decrease in glycosides, but was delayed. The phenolic response of Norway-spruce phloem to inoculation is modulated by the presence of the fungus and is focused on the formation of insoluble compounds.     

Induction of phenylalanine ammonia lyase activity and control of Sphaeropsis sapinea infection in Pinus radiata by 5-chlorosalicylic acid

Phenylalanine ammonia lyase (PAL) activity increased rapidly in Pinus radiata seedlings after treatment with 2 mm 5-chlorosalicylic acid (5CSA) and peaked after 48 h at four-times the level of the control. Elevation of PAF is often associated with the induction of host resistance mechanisms. Thus, effects of 5CSA on subsequent infection of P. radiata by Sphaeropsis sapinea was investigated. Only one out of 16 seedlings treated with 2 mm 5CSA, 4 days before inoculation, developed deadtop compared to 14 of the controls. The 5CSA was directly inhibitory at this concentration and reduced growth rate of S. sapinea on agar by 27%. However, antifungal activity does not fully account for the observed disease control afforded by 5CSA since 0.5 mm and 0.25 mm 5CSA, which did not inhibit growth of S. sapinea, significantly reduced the incidence of deadtop. Furthermore, 1 mm 5CSA protected seedlings against fresh inoculation challenge for up to 32 days after application. Such durable resistance is unlikely to be attributable to the direct effects of the applied compound and may suggest the involvement of inducible host resistance mechanisms.     

Protocol for screening rust disease resistance in Dalbergia sissoo

A protocol has been developed for inoculation of rust disease in Dalbergia sissoo using urediniospores of Maravilia achroa. This is the first time that disease symptoms caused by M. achroa were artificially produced. The method has been successfully employed for screening 15 clones of D. sissoo against rust disease. Clone S‐167 was the most resistant remaining free from infection. Clones S‐57, S‐106 and S‐124 were also resistant. Clones S‐19, S‐24 and S‐89 were susceptible.