Gall Size Research Articles

The Gall Wasp Ophelimus migdanorum and Its Parasitoid Closterocerus chamaeleon on Eucalyptus globulus at Two Sites with Different Rainfall Parameters in Bogotá, Colombia.

As a pest, the gall wasp Ophelimus migdanorum poses a risk to several Eucalyptus species in Colombia. In the tropical Andes, its biological development and the damage it causes can be influenced by climate, particularly rainfall. In this regard, we examined gall phenology, population fluctuation, and leaf damage caused by O. migdanorum, and its parasitoid Closterocerus chamaeleon, over 5months at two sites with contrasting rainfall in peri-urban areas of Bogotá, Colombia. Gall phenology and foliar damage were assessed on 10 trees per site. We characterized gall phenology by assessing their size and color, wasps' developmental stages, as well as affected leaf area and gall density on the leaf blade. Additionally, the individuals found in five attraction traps at each site were quantified biweekly to record population fluctuations. The effect of rainfall on wasp frequency and gall density between sampling sites and dates was compared using the chi-square test, while the relationship with rainfall was evaluated using the Kruskal-Wallis test. Only females of the gall wasp and its parasitoid were observed during the study, displaying multivoltine behavior. Six developmental stages of the galls were differentiated, each characterized by distinct coloration. Drier microclimates favored gall size, wasps' development stage frequency, population trend, and foliar damage. The general trend indicated a greater abundance of the parasitoid C. chamaeleon compared to O. migdanorum. Our study suggests that O. migdanorum has a less damaging effect on E. globulus in areas with rainy microclimates.

Changes in primary metabolism and associated gene expression during host-pathogen interaction in clubroot resistance of Brassica napus.

The role of primary metabolism during Brassica napus-Plasmodiophora brassicae interaction leading to clubroot resistance has not yet been investigated thoroughly. In this study, we investigated some of the primary metabolites and their derivatives as well as expression of the genes involved in their biosynthesis to decipher this host-pathogen interaction. For this, two sets (clubroot resistant and susceptible) of canola lines were inoculated with P. brassicae pathotype 3A to investigate the endogenous levels of primary metabolites at 7-, 14-, and 21-days after inoculation (DAI). The associated pathways were curated, and expression of the selected genes was analyzed using qRT-PCR. Our results suggested the possible involvement of polyamines (spermidine and spermine) in clubroot susceptibility. Some of the amino acids were highly abundant at 7- or 14-DAI in both resistant and susceptible lines; however, glutamine and the amino acid derivative phenylethylamine showed higher endogenous levels in the resistant lines at later stages of infection. Organic acids such as malic, fumaric, succinic, lactic and citric acids were abundant in the susceptible lines. Conversely, the abundance of salicylic acid (SA) and the expression of benzoate/salicylate carboxyl methyltransferase (BSMT) were higher in the resistant lines at the secondary stage of infection. A reduced disease severity index and gall size were observed when exogenous SA (1.0 mM) was applied to susceptible B. napus; this further supported the role of SA in clubroot resistance. In addition, a higher accumulation of fatty acids and significant upregulation of the pathway genes, glycerol-3-phosphate dehydrogenase (GPD) and amino alcohol phosphotransferase (AAPT) were observed in the resistant lines at 14- and 21-DAI. In contrast, some of the fatty acid derivatives such as phosphatidylcholines represented a lower level in the resistant lines. In conclusion, our findings provided additional insights into the possible involvement of primary metabolites and their derivatives in clubroot resistance.

Utilizing endophytic plant growth-promoting bacteria and the nematophagous fungus Purpureocillium lilacinum as biocontrol agents against the root-knot nematode (Meloidogyne incognita) on tomato plants

AbstractThe present investigation was designed to assess how administering biocontrol agents (BCAs) made from the nematophagous endophytic fungus MR20 (Purpureocillium lilacinum) or a mixture of endophytic plant growth-promoting bacteria MR12 (Pseudomonas fluorescens), and MR25 (Serratia marcescens), could enhance tomato growth and resistance to Meloidogyne incognita under in vivo conditions. The three strains’ cell-free culture filtrates showed a strong nematocidal impact (P < 0.05) on M. incognita infective second-stage juveniles (J2s). The highest mortality rates by the three BCAs were at concentrations of 80%, followed by 60%, 40%, and 20%. The in vitro hatching of free eggs was found to be considerably (P < 0.05) reduced as the concentrations of the endophytic bacteria MR12, and MR25 were increased in the order 1 × 105, 1 × 106, 1 × 107, 0.5 × 108, to 1 × 108 colony forming units (CFU)/mL, and the maximum nematicidal activity in killing M. incognita free eggs occurred at 1 × 108 CFU/mL. A statistically significant (P < 0.05) reduction in the percentage of M. incognita-free eggs retrieved was seen in vitro when various concentrations of the endophytic fungus MR20 were applied compared to the negative control (distilled water). The endophytic fungus MR20 had the highest nematicidal activity against M. incognita free eggs at a concentration of 3 × 106 CFU/mL. The application of P. lilacinum or a combination of P. fluorescens and S. marcescens to tomato plants in the presence of M. incognita under greenhouse conditions resulted in a significant increase (P < 0.05) in root and shoot fresh weight, number of leaves, weight of leaves, and stem diameter when compared to the positive control treatment that contained only M. incognita. Treatment with P. lilacinum was more effective (P < 0.05) than P. fluorescens and S. marcescens in reducing egg masses per root, J2s per 100 g of soil, egg masses per 100 g of soil, J2s + eggs of M. incognita per 100 g of soil, reproduction factor, and reduction percentage after 60 days under greenhouse conditions. Nevertheless, the reduction of M. incognita gall size categorization (> 4 mm, 2–4 mm, and < 2 mm) was more effectively achieved by treatment with P. fluorescens and S. marcescens than by treatment with P. lilacinum. Under in vivo conditions, the tested P. lilacinum or a mixture of P. fluorescens and S. marcescens effectively controlled nematode population densities below the economic threshold.

Successful gall induction on wild roses by gall wasps under lab conditions

Abstract Plant galls are unique outgrowths caused by various organisms, including insects, serving as nourishment for the inducer's larvae. Despite the taxonomists and ecologists attempts to elucidate the mechanisms behind plant gall formation, its understanding is still incomplete. Modern genetic techniques allow in‐depth analysis of the molecular processes, but variations across species entangle the analysis. Establishing laboratory‐friendly plant–gall inducer communities is crucial, yet past attempts have faced challenges. Our study partly aimed to create a laboratory community involving wild roses (Rosa sp.) and rose gall wasps belonging to the genus Diplolepis as gall‐inducing insects. Controlled indoor conditions were optimized for plant growth. Wild roses were transplanted, then exposed to gall inducers, and monitored. Successfully initialized gall growth was measured and analysed, revealing insights into the impact of plant vigour on gall size as a secondary aim. Our study successfully established a novel laboratory community for further research on gall formation mechanisms.

The cynipid gall wasp Diplolepis rosae is more successful in North America than in Europe because of enemy release

Abstract The Enemy Release Hypothesis predicts that introduced species in their new range are freed from natural enemies (e.g., pathogens, parasitoids and predators) that control their populations. Diplolepis rosae (Hymenoptera, Diplolepididae), native to the Western Palearctic, induces readily apparent galls on wild roses (Rosa spp.) that support a robust component community of inquilines, parasitoids and hyperparasitoids in its native range. D. rosae was introduced to North America in the mid‐1800s, and has since become widespread and common across the continent. We compared the insect communities associated with D. rosae galls from Canada and the US Pacific Northwest with those of Eastern Europe. Throughout its introduced range, parasitism rates were lower compared with galls in their natural range. Component communities were also less diverse and species‐rich. The relationship between gall size and parasitism rates showed no significant difference between the two continents. These results show that the component community in its introduced range is depauperate and provide support for the Enemy Release Hypothesis.

Cynipid wasps systematically reprogram host metabolism and restructure cell walls in developing galls.

Many insects have evolved the ability to manipulate plant growth to generate extraordinary structures called galls, in which insect larva can develop while being sheltered and feeding on the plant. In particular, cynipid (Hymenoptera: Cynipidae) wasps have evolved to form morphologically complex galls and generate an astonishing array of gall shapes, colors, and sizes. However, the biochemical basis underlying these remarkable cellular and developmental transformations remains poorly understood. A key determinant in plant cellular development is cell wall deposition that dictates the physical form and physiological function of newly developing cells, tissues, and organs. However, it is unclear to what degree cell walls are restructured to initiate and support the formation of new gall tissue. Here, we characterize the molecular alterations underlying gall development using a combination of metabolomic, histological, and biochemical techniques to elucidate how valley oak (Quercus lobata) leaf cells are reprogrammed to form galls. Strikingly, gall development involves an exceptionally coordinated spatial deposition of lignin and xylan to form de novo gall vasculature. Our results highlight how cynipid wasps can radically change the metabolite profile and restructure the cell wall to enable the formation of galls, providing insights into the mechanism of gall induction and the extent to which plants can be entirely reprogrammed to form unique structures and organs.

The relative infection potential of Meloidogyne incognita and M. graminicola in the basmati rice cultivar PB1121

AbstractRoot‐knot nematodes, Meloidogyne spp., are considered one of the major biotic constraints for global rice production. Although the role of Meloidogyne graminicola in root‐knot disease progression is well investigated, negligible research has been performed to understand the damaging potential of M. incognita in rice. In this study, the comparative infection biology of M. incognita and M. graminicola was investigated in the basmati rice cultivar PB1121 growing in Pluronic gel medium and soil. Intriguingly, M. incognita laid eggs outside the galled root, whereas M. graminicola egg masses were deposited inside the galled root. This differential egg‐laying pattern can be correlated with their specific adaptations in irrigated and upland rice production systems. Although M. incognita induced a greater number of galls than M. graminicola, M. graminicola gall sizes were comparatively larger than those of M. incognita. The greater reproductive success of M. graminicola than M. incognita did not cause any difference in their yield reduction potential in PB1121. PB1121 was equally vulnerable to M. incognita and M. graminicola in terms of reduction in plant height, tiller numbers per plant and grain yield per plant. Our research suggests that M. incognita also causes economic damage to rice, and nematode management tactics must consider the damaging potential of both nematode species.

Epidermal and subepidermal changes during the formation of hairy galls induced by Eriophyidae on Avicennia schaueriana leaves.

Leaf-galling Eriophyidae (Acarina) may promote simple or complex alterations in the organs of their host plants, such as an increase in indumentum density or the reorganization of epidermis and ground system tissue patterns. To test if hairy galls of Eriophyidae on Avicennia schaueriana (Acanthaceae) are related to complex changes, leaf galls in distinct developmental phases were compared to non-galled leaves using anatomical, histochemical, and histometric analyses. Quantitative comparisons of preferential gall induction sites and gall area according to distinct leaf portions were made to evaluate if the impacts of gall formation can be related to the distinct potentialities of leaf microsites. The apical portion of the leaves and leaf margins were the sites with the highest occurrence of galls, but no relationship was detected between gall area and induction site. The gall anatomy revealed that epidermal features are influenced the most with the development of abnormal stomata and projected or sunken salt glands. The most striking change is the neoformation of elongated filiform trichomes on the abaxial surface (where the mites occur) that accumulate reducing sugars and proteins. The filiform trichomes may protect the inducers against abiotic stressors and enemies, and the primary metabolites that accumulate are important foods for mites. The mesophyll has simple alterations, only in the spongy parenchyma. Complex alterations occur only in abaxial epidermal cells close to feeding sites of the inducer. The number of inducers per gall seems to be the most important influence on gall size, since gall area is not related to the position in the leaves.

Fine-Scale analysis of both wild and cultivated horned galls provides insight into their quality differentiation

BackgroundGalla chinensis is a traditional Chinese medicine (TCM) produced due to the interaction between the Fordinae aphids and the Rhus plant species. Horned galls with high tannin content are the most widely cultivated gall type, and Wufeng county of Hubei province in China is the center of cultivation. However, long-term artificial cultivation and domestication of horned galls to meet the increasing production demand have led to quality degradation. Understanding the reasons underlying quality degradation is urgent for horned gall production and application. The present study used a combination of metabolic, genetic, and ecological analyses to investigate the quality and genetic differentiation of the horned galls under long-term domestication as well as the potential relationships between them.ResultsAnalysis of gallic acid content and other three phenotypic traits (fresh weight, gall size, and wall thickness) revealed quality differentiation of horned galls collected from five locations in Wufeng, in which the cultivated samples from Wang Jiaping (WJP) showed the highest degradation. Genetic differentiation between the cultivated and wild Rhus chinensis trees in WJP, and between WJP and the other populations was detected based on SSR molecular markers, however, no significant difference in genetic structure was seen for the aphid populations. Among the various ecological factors examined, temperature was identified as the primary one affecting the quality of horned galls.ConclusionsBoth genetic and ecological factors caused quality differentiation of horned galls. The collection of diverse germplasm of host trees and aphids will help reduce the quality degradation of horned galls in Wufeng.

Biology and morphometric relationship of gall inducers Contarinia sp. and corresponding parasitoids for swollen galls of Nitraria sibirica pall.

Galls function as provide shelter for gall inducers, guarding them against their natural enemies. Previous research has illuminated the interactions between galls, gall inducers, and their corresponding parasitoids within various caltrop plants. However, less is known about these relationships within Nitraria sibirica, particularly regarding the efficacy of parasitism. Therefore, this study aimed to identify the morphometric relationships among the swollen galls, gall inducers, and their parasitoids. Two species of gall inducers and three species of parasitoids were obtained from the swollen galls of N. sibirica. The correlations of the parasitization indexes, the lifespan of gall inhabitants, and temperature and the morphometric relationships between the galls and their inhabitants were analyzed. The dominant gall inducer identified was Contarinia sp. (Diptera: Cecidomyiidae). Furthermore, it was observed that three solitary parasitoids attacked Contarinia sp. in the swollen galls, with only Eupelmus gelechiphagus acting as an idiobiont ectoparasitoid. The dominant parasitoids were Platygaster sp. and Cheiloneurus elegans at sites 1 and 2, respectively, with Platygaster sp. displaying greater abundance than C. elegans in the swollen galls. The lifespan of the gall inhabitants shortened gradually as the temperature increased. Moreover, the optimal number of gall chambers ranged from two to four per swollen gall with maximized fitness, which can be considered the optimal population density for the gall inducer Contarinia sp. Morphometric analysis exhibited a strong linear correlation between gall size and chamber number or the number of gall inhabitants, as well as a weak correlation between gall size and body size of the primary inhabitants of swollen galls. Our results highlight the importance of the biological investigation of parasitoids and gall inducers living in closed galls with multiple chambers and may pave the way for potential application in biological control.

Molecular Aspects of Gall Formation Induced by Mites and Insects.

Recent publications on gall formation induced on the leaves of dicotyledonous flowering plants by eriophyoid mites (Eriophyoidea) and representatives of four insect orders (Diptera, Hemiptera, Hymenoptera, Lepidoptera) are analyzed. Cellular and molecular level data on the stimuli that induce and sustain the development of both mite and insect galls, the expression of host plant genes during gallogenesis, and the effects of these galling arthropods on photosynthesis are considered. A hypothesis is proposed for the relationship between the size of galls and the volume of secretions injected by a parasite. Multistep, varying patterns of plant gene expression and accompanying histo-morphological changes in the transformed gall tissues are apparent. The main obstacle to better elucidating the nature of the induction of gallogenesis is the impossibility of collecting a sufficient amount of saliva for analysis, which is especially important in the case of microscopic eriophyoids. The use of modern omics technologies at the organismal level has revealed a spectrum of genetic mechanisms of gall formation at the molecular level but has not yet answered the questions regarding the nature of gall-inducing agents and the features of events occurring in plant cells at the very beginning of gall growth.

Selection of cowpea (Vigna unguiculata [L.] Walp) accessions resistant to root‐knot nematode

AbstractCowpea, Vigna unguiculata, is a legume adapted to regions with a tropical climate and limited water resources, and it has become a basic food source and income generator. Root‐knot nematodes (Meloidogyne spp.) cause substantial damage to the root system, resulting in yield losses. Genetic resistance is one of the most efficient management methods. The objective of this study was to generate information on genetic resistance to Meloidogyne incognita and Meloidogyne javanica of Brazilian cowpea genotypes. For this, 18 accessions of cowpea from the collection of Embrapa Meio‐Norte and six control accessions of cowpea were tested in completely randomized design with six replications, evaluated 60 days after inoculation, and carried out twice in time. To assess root penetration of nematodes and root gall formation in NIL‐CB3‐isoline, which allows reproduction but limits gall formation, nematodes inside the roots were stained with acid fuchsin, after inoculation with 5000 J2, with estimates at 72 h time‐points up to 28 days. None of the 18 accessions from Embrapa showed resistance to M. javanica, but accessions MNC06‐895‐1, MNC04‐14, MNC06‐909‐6, 98, MNCO4‐907‐35, and Am60‐15 showed resistance to M. incognita. There was a reduction in the number and size of galls in the NIL‐CB3 egg isoline, and an increase in formation of males, which are not important for parasitism. Given the importance and prospects for expansion of the cowpea crop throughout Brazil, the results of this study contribute to the knowledge on the reaction of Brazilian cowpea genotypes to the root‐knot nematode.

A quantitative and qualitative assessment of sugar beet genotype resistance to root-knot nematode, Meloidogyne incognita.

Sugar beet productivity is highly constrained by the root-knot nematode (RKN) Meloidogyne incognita. Eight sugar beet genotypes were screened under greenhouse conditions for their susceptibility to M. incognita according to an adapted quantitative scheme for assignment Canto-Saenz's host suitability (resistance) designations (AQSCS). Besides, the degree of susceptibility or tolerance of the examined genotypes was recorded by the modified host-parasite index (MHPI) scale based on yield performance. In addition, single nucleotide polymorphism (SNP) was also determined. Sugar beet genotypes have been classified into four categories for their susceptibility or tolerance according to the AQSCS scale. The first category, the moderately resistant (MR) group implies only one variety named SVH 2015, which did not support nematode reproduction (RF≤1), and had less root damage (GI≈2). Second, the tolerant group (T) involving Lilly and Halawa KWS supported fairly high nematode reproduction (RF>1) with relatively plant damage (GI≤2). Whereas the susceptible (S) category involved four varieties, FARIDA, Lammia KWS, Polat, and Capella, which supported nematode reproduction factor (RF>1) with high plant damage (GI>2). The fourth category refers to the highly susceptible (HYS) varieties such as Natura KWS that showed (RF≤1) and very high plant damage (GI>2). However, the MHPI scale showed that Lammia KWS variety was shifted from the (S) category to the (T) category. Results revealed significant differences among genotypes regarding disease severity, yield production, and quality traits. The SVH 2015 variety exhibited the lowest disease index values concerning population density with 800/250 cm3 soils, RF=2, root damage/gall index (GI=1.8), gall size (GS=2.3), gall area (GA=3.7), damage index (DI=3.4), susceptibility rate (SR=2.4), and MHP index (MHPI=2.5). However, Lammia KWS showed the highest disease index values regarding population density with 8890/250 cm3 soils, RF= 22.2, GI= 4.8, and SR= 14.1. Meanwhile, Natura KWS the highest GS, GA and MHPI with 7.1, 8 and 20.9, respectively. The lowest DI was achieved by Capella (DI= 6) followed by Lammia KWS (DI= 5.9). For yield production, and quality traits, SVH 2015 exhibited the lowest reductions of sugar yields/beet's root with 11.1%. While Natura KWS had the highest reduction with 79.3%, as well as it showed the highest reduction in quality traits; including sucrose, T.S.S, and purity with 65, 27.3, and 51.9%, respectively. The amino acid alignment and prediction of the DNA sequences revealed the presence of five SNPs among all sugar beet verities.

Pest characteristics of Trigonorhinus sp. (Coleoptera: Anthribidae) and the developmental process of its galls in Caragana liouana Zhao Y. Chang and Yakovlev

Abstract In recent years, a Trigonorhinus sp. (Coleoptera) has caused serious damage to Caragana liouana Zhao Y. Chang and Yakovlev, a major ecological restoration shrub in China. Here, we survey the occurrence and damage pattern of Trigonorhinus sp. and its galls and compare the growth of affected and unaffected plants. Trigonorhinus sp. larvae usually infest the main trunk and lateral branches of the plant, causing the affected branches to become partially swollen and verrucose. Galls develop in stages depending on whether eggs are laid in May or July and proceed in sequence over three years from egg-laying to formation, expansion, dormancy, maturity, dormancy, maturity, and death. Galls inhibit plant development to some extent. On average, six (at least one, and no more than 18; standard error of the mean = 3) larvae occupy each gall, and the number of larvae within a gall did not significantly affect gall size. Gall size significantly affected branch dieback, and large-diameter infested branches had larger galls. This study clarifies the growth dynamics of Trigonorhinus sp. galls and provides a basis for further research into the growth mechanism of the species’ galls.

Mechanisms of hydraulic conductivity in the leaf galls of Meunieriella sp. (Cecidomyiidae) in Avicennia schaueriana (Acanthaceae): does vascularization explain the preferred sites of induction?

Intralaminar galls of Meunieriella result from ground tissue proliferation in leaves of Avicennia schaueriana, a typical halophytic mangrove. We tested if the preferred sites of gall induction were the midribs and secondary veins (SV) at the basal leaf portion, where the galls were expected to be largest; and if the vascular system in galls and adjacent regions was altered to favour water supply in galls, thus increasing their growth. Gall induction sites and gall sizes were quantified according to leaf portions and regions. Anatomical and histometric analyses in vascular and ground tissues of galls and adjacent regions were compared to equivalent regions of non-galled leaves. The galls were largest at basal sites on leaves, the midrib and SV. More galls occurred on the apical portion of the leaf, and on the leaf blade and secondary vein regions. Changes in shape and vascular system area, number and diameter of vessel elements were detected in both galls and adjacent regions. Fewer and smaller-sized vessel elements were observed in regions proximal to the galls and inside them. Gall size is not related with preferred induction sites, which could be explained by factors such as thermal balance. Alterations in the vascular system indicate reduced hydraulic conductivity in the xylem in the proximal region and inside galls, leading to water leakage to gall parenchyma cells. This compensatory mechanism explains the expansion and proliferation of water storage and spongy parenchyma cells in the galls, explaining the higher growth in more vascularized regions.

Local auxin synthesis mediated by YUCCA4 induced during root-knot nematode infection positively regulates gall growth and nematode development.

Parasites and pathogens are known to manipulate the host's endogenous signaling pathways to facilitate the infection process. In particular, plant-parasitic root-knot nematodes (RKN) are known to elicit auxin response at the infection sites, to aid the development of root galls as feeding sites for the parasites. Here we describe the role of local auxin synthesis induced during RKN infection. Exogenous application of auxin synthesis inhibitors decreased RKN gall formation rates, gall size and auxin response in galls, while auxin and auxin analogues produced the opposite effects, re-enforcing the notion that auxin positively regulates RKN gall formation. Among the auxin biosynthesis enzymes, YUCCA4 (YUC4) was found to be dramatically up-regulated during RKN infection, suggesting it may be a major contributor to the auxin accumulation during gall formation. However, yuc4-1 showed only very transient decrease in gall auxin levels and did not show significant changes in RKN infection rates, implying the loss of YUC4 is likely compensated by other auxin sources. Nevertheless, yuc4-1 plants produced significantly smaller galls with fewer mature females and egg masses, confirming that auxin synthesized by YUC4 is required for proper gall formation and RKN development within. Interestingly, YUC4 promoter was also activated during cyst nematode infection. These lines of evidence imply auxin biosynthesis from multiple sources, one of them being YUC4, is induced upon plant endoparasitic nematode invasion and likely contribute to their infections. The coordination of these different auxins adds another layer of complexity of hormonal regulations during plant parasitic nematode interaction.

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to <em>Plasmodiophora brassicae</em>

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae.

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.

Phytohormones in galls on eucalypt trees and in the gall‐forming wasp Leptocybe invasa (Hymenoptera: Eulophidae)

Abstract The role played by phytohormones in insect galls and the mechanisms underlying plant manipulation by gall makers are still unclear. Galls made by Leptocybe invasa on two Eucalyptus species were sampled, the correlations between the levels of nine phytohormones in galls and gall size were analysed, and phytohormone levels in the interior and exterior tissues of galls and L. invasa were determined. During two larval stages, the levels of three forms of cytokinins (CKs) in the galls of the two plant species all showed significantly positive correlations with gall size. Brassinosteroids (BRs) and dihydrozeatin riboside (DHZR) occurred at significantly higher levels in the interior tissues of the galls compared to in the exterior tissues of galls. Isopentenyl adenosine (IPA) was significantly higher in L. invasa larvae themselves, compared to levels in the interior and exterior tissues of galls. However, the levels of DHZR and BR were significantly lower or similar in the L. invasa pupae compared to that in the interior and exterior tissues of the galls. These findings indicate that phytohormones, especially CKs, act as key regulators of L. invasa galls, and that the larvae of L. invasa may themselves be a source of CKs.

Reduction of root-knot nematode infection with compost and nematode-trapping fungus in greenhouse setting

Arthrobotrys oligospora is a nematode-trapping fungus and one of the most extensively researched biological control agents of plant-parasitic nematodes. Urea and composted organic matter (COM) may also control nematodes by making rhizospheric environment more favorable to beneficial microbes and also boosted the plant growth improvement. Thus, combining A. oligospora with urea and COM may improve nematode biocontrol. For this. an in vitro experiment was performed with three replicates of each parameter and repeated thrice. Inoculation with M. incognita in culture plates revealed that the fungus creates adhesive traps comprised of hyphae loops within 24 h. However, maximal capture was observed after 7 days of incubation. Furthermore, the A. oligospora culture filtrate was highly poisonous to the nematodes. Green house trial was conducted complete randomized block design with three replicates of each treatment, including the control and inoculated control pots. The plants treated with A. oligospora alone or in combination with urea and COM showed improved growth, yield and biochemical characteristics, when compared to M. incognita inoculated plants. Antioxidant enzyme activity (POD, SOD, CAT and PAL) was substantially increased in the plants treated with A. oligospora in combination with urea and COM. Maximum and significant decrease in number of gall (86.89%), size of gall (76.52%), number of egg masses (88.86%) and disease index were observed when fungus was used in combination with COM. Histological examination showed that the presence of A. oligopsora reduced nematode penetration and feeding site when compared to roots infected with only M. incognita, which show breakdown of the vascular bundle due to nematode feeding. These findings imply that combining A. oligospora with urea and COM has the ability to promote plant resilience as well as more trap formation by fungus resulted in prevent M. incognita infection and serve as an environmentally safe alternative to chemical nematicides.