Histopathology of the Plasmodiophora brassicae-Chinese Cabbage Interaction in Hosts Carrying Different Sources of Resistance

Clubroot is a serious soil-borne disease of crucifers caused by the obligate parasite Plasmodiophora brassicae. The genetic basis and histopathology of clubroot resistance in two Chinese cabbage (Brassica rapa ssp. pekinensis) inbred lines Bap055 and Bap246, challenged with pathotype 4 of P. brassicae, was evaluated. The Chinese cabbage cultivar “Juxin” served as a susceptible check. The resistance in Bap055 was found to be controlled by the CRa gene, while resistance in Bap246 fit a model of control by unknown recessive gene. Infection of the roots by P. brassicae was examined by inverted microscopy. Despite their resistance, primary and secondary infection were observed to occur in Bap055 and Bap246. Primary infection was detected at 2 days post-inoculation (DPI) in “Juxin,” at 4 DPI in Bap055, and at 6 DPI in Bap246. Infection occurred most quickly on “Juxin,” with 60% of the root hairs infected at 10 DPI, followed by Bap055 (31% of the root hairs infected at 12 DPI) and Bap246 (20% of the root hairs infected at 14 DPI). Secondary infection of “Juxin” was first observed at 8 DPI, while in Bap055 and Bap246, secondary infection was first observed at 10 DPI. At 14 DPI, the percentage of cortical infection in “Juxin,” Bap055 and Bap246 was 93.3, 20.0, and 11.1%, respectively. Although cortical infection was more widespread in Bap055 than in Bap246, secondary infection in both of these hosts was restricted relative to the susceptible check, and the vascular system remained intact. A large number of binucleate secondary plasmodia were observed in “Juxin” and the vascular system was disrupted at 16 DPI; in Bap055 and Bap246, only a few secondary plasmodia were visible, with no binucleate secondary plasmodia. The defense mechanisms and expression of resistance appears to differ between Chinese cabbage cultivars carrying different sources of resistance.

Transcriptome and Coexpression Network Analyses Reveal Hub Genes in Chinese Cabbage (Brassica rapa L. ssp. pekinensis) During Different Stages of Plasmodiophora brassicae Infection

Clubroot, caused by the soil-borne protist Plasmodiophora brassicae, is one of the most destructive diseases of Chinese cabbage worldwide. However, the clubroot resistance mechanisms remain unclear. In this study, in both clubroot-resistant (DH40R) and clubroot-susceptible (DH199S) Chinese cabbage lines, the primary (root hair infection) and secondary (cortical infection) infection stages started 2 and 5 days after inoculation (dai), respectively. With the extension of the infection time, cortical infection was blocked and complete P. brassica resistance was observed in DH40R, while disease scales of 1, 2, and 3 were observed at 8, 13, and 22 dai in DH199S. Transcriptome analysis at 0, 2, 5, 8, 13, and 22 dai identified 5,750 relative DEGs (rDEGs) between DH40R and DH199S. The results indicated that genes associated with auxin, PR, disease resistance proteins, oxidative stress, and WRKY and MYB transcription factors were involved in clubroot resistance regulation. In addition, weighted gene coexpression network analysis (WGCNA) identified three of the modules whose functions were highly associated with clubroot-resistant, including ten hub genes related to clubroot resistance (ARF2, EDR1, LOX4, NHL3, NHL13, NAC29, two AOP1, EARLI 1, and POD56). These results provide valuable information for better understanding the molecular regulatory mechanism of Chinese cabbage clubroot resistance.

The Role of Primary and Secondary Infection in Host Response to Plasmodiophora brassicae

The disease cycle of Plasmodiophora brassicae consists of a primary phase in root hairs followed by a secondary phase in the root cortex and adjacent tissues. However, the role of root hair infection in subsequent cortical infection and development of P. brassicae is not well understood. To examine the role of the primary and secondary stages separately, inoculation studies with resting spores (source of primary zoospores) and secondary zoospores of a virulent and avirulent pathotype were conducted on canola (Brassica napus). The size of secondary zoospores and number of nuclei were also examined. The zoospores were larger (≈9.6 to 14.4 μm) than in previous reports and all were uninucleate. Inoculation with secondary zoospores alone produced both primary and secondary infection, even with the avirulent pathotype. No symptoms developed from inoculation with avirulent primary zoospores but tiny, bead-shaped clubs developed from inoculation with avirulent secondary zoospores. Inoculation with virulent secondary zoospores alone resulted in lower disease severity than inoculation with virulent resting spores alone. The results indicate that recognition of infection by the host and initiation of a response (induction or suppression of resistance) occurs during primary infection, although recognition can also occur during cortical infection and development.

Effect of Temperature on Cortical Infection by Plasmodiophora brassicae and Clubroot Severity

A study was conducted to assess the effect of temperature on infection and development of Plasmodiophora brassicae in the root cortex of Shanghai pak choy (Brassica rapa subsp. chinensis) and on subsequent clubroot severity. Ten-day-old seedlings were grown individually, inoculated with resting spores, and maintained in growth cabinets at 10, 15, 20, 25, and 30°C. Seedlings were harvested at 2-day intervals, starting 8 days after inoculation (DAI) and continuing until 42 DAI. Roots were assessed at 4-day intervals for the incidence of cortical infection and stage of infection (young plasmodia, mature plasmodia, and resting spores), at 2-day intervals for symptom development and clubroot severity, and at 8-day intervals for the number of spores per gram of gall. Temperature affected every stage of clubroot development. Cortical infection was highest and symptoms were observed earliest at 25°C, intermediate at 20 and 30°C, and lowest and latest at 15°C. No cortical infection or symptoms were observed at 42 DAI in plants grown at 10°C. A substantial delay in the development of the pathogen was observed at 15°C. Resting spores were first observed at 38 DAI in plants at 15°C, 26 DAI at 20 and 30°C, and 22 DAI at 25°C. The yield of resting spores from galls was higher in galls that developed at 20 to 30°C than those that developed at 15°C over 42 days of assessment. These results support the observation in companion studies that cool temperatures result in slower development of clubroot symptoms in brassica crops, and demonstrate that the temperature has a consistent pattern of effect throughout the life cycle of the pathogen.

Roles for Azorhizobial Nod Factors and Surface Polysaccharides in Intercellular Invasion and Nodule Penetration, Respectively

In the symbiotic interaction between Azorhizobium caulinodans and Sesbania rostrata root and stem-borne nodules are formed. The bacteria enter the host via intercellular spaces at lateral or adventitious root bases and form infection pockets in outer cortical layers. Infection threads guide the bacteria to nodule primordia where plant cells are invaded. To identify bacterial functions that are required for this infection process, two mutants defective in nodulation were studied; one produced no Nod factors (nodA mutant), the other had altered surface polysaccharides (SPS) and induced the formation of pseudo-nodules. Bacteria were visualized with the help of a nodA-uidA reporter fusion that was functional during nodule development and in bacteroids. In contrast to the SPS mutant, nodA mutants were unable to colonize outer cortical regions. In mixed inoculations with both mutants, functional nodules were formed, the central tissue of which was occupied by the nodA mutant. These observations suggest that SPS play a role in deeper invasion and that Nod factors are necessary for entry. Simultaneous application of purified Nod factors and nodA mutant bacteria restored the formation of outer cortical infection pockets leading to the conclusion that intercellular infection is an active process that is dependent on bacterial Nod factor signaling.

Microscopy of Fusarium solani infected rough lemon citrus fibrous roots

Mode of Fusarium solani penetration into rough lemon citrus rootstock seedling fibrous roots was determined and root symptomatology was evaluated using light, scanning, and transmission electron microscopy. Roots contained limited subepidermal infection 15 h after inoculation. Invasion of epidermal cells was principally through broken epidermal and root hair cell walls, but direct penetration also occurred. Cortical infection was primarily intracellular. Severe root rot was present 36–60 h after inoculation and in most cases infection after 60 h resulted in disruption of the phloem and endodermis around the still relatively intact primary xylem. Naturally infected roots from field trees were collected in an advanced stage of rot. In these, F. solani digestion of middle lamellae of primary xylem was common, and infection and erosion of secondary vessel element walls occurred frequently.

Histopathology of Pythium infected strawberry roots

Host–parasite interactions between eight Pythium species, previously isolated from necrotic strawberry root tissue, and Fragaria × ananassa Duch. runner plant main roots were studied. When excised roots were placed in water on petri dish cultures, P. rostratum and P. hypogenum infection was limited to the root tip epidermis. Pythium irregulare, P. perniciosum, P. dissotocum, P. sylvaticum, P. ultimum, and unidentified isolate 1E penetrated the epidermis several centimeters along the zone of maturation. Penetration occurred most frequently through root hairs by hyphae; resulting infections usually stopped in the epidermis or adjacent cortex, and here hyphae formed sporangium-like structures, oogonia, and oospores. Extensive cortical infection by hyphae was primarily limited to the zone of elongation. Extent of epidermal and cortical infection in roots grown in soil with P. irregulare, P. dissotocum, and P. sylvaticum was similar to petri dish test results. Stelar infection by P. sylvaticum and P. dissotocum occurred mainly in phloem cells, but did not advance far into the region of maturation. Numerous oogonia formed in infected steles and usually in adjacent cortical cells. Infected roots ranged from a white to grey, water-soaked color. Extensively infected root tips typically were unhealthy, succulent, and thin.