Co-Inoculation with Rhizobia and AMF Inhibited Soybean Red Crown Rot: From Field Study to Plant Defense-Related Gene Expression Analysis

Background: Soybean red crown rot is a major soil-borne disease all over the world, which severely affects soybean production. Efficient and sustainable methods are strongly desired to control the soil-borne diseases. Principal Findings: We firstly investigated the disease incidence and index of soybean red crown rot under different phosphorus (P) additions in field and found that the natural inoculation of rhizobia and arbuscular mycorrhizal fungi (AMF) could affect soybean red crown rot, particularly without P addition. Further studies in sand culture experiments showed that inoculation with rhizobia or AMF significantly decreased severity and incidence of soybean red crown rot, especially for coinoculation with rhizobia and AMF at low P. The root colony forming unit (CFU) decreased over 50 % when inoculated by rhizobia and/or AMF at low P. However, P addition only enhanced CFU when inoculated with AMF. Furthermore, root exudates of soybean inoculated with rhizobia and/or AMF significantly inhibited pathogen growth and reproduction. Quantitative RT-PCR results indicated that the transcripts of the most tested pathogen defense-related (PR) genes in roots were significantly increased by rhizobium and/or AMF inoculation. Among them, PR2, PR3, PR4 and PR10 reached the highest level with co-inoculation of rhizobium and AMF. Conclusions: Our results indicated that inoculation with rhizobia and AMF could directly inhibit pathogen growth and reproduction, and activate the plant overall defense system through increasing PR gene expressions. Combined wit

Resistance to Septoria Musiva in Hybrid Poplar

Septoria leaf spot and stem canker, caused by the fungal pathogen Mycosphaerella populorum Thompson (Anamomorph = Septoria musiva Peck). An efficient greenhouse disease resistance screening is essential for the development of resistant clones. Fourteen clones of hybrid poplar were inoculated with spore suspension. A regression model with parameters (lesion number and proportion necrotic area) is consistently and accurately predicted field resistance categories of the most resistant and susceptible clones. In second experiments, the infection biology of S. musiva was examined at several time points by scanning electron microscopy (6 h, 12 h, 24 h, 72 h, 1-week and 3-week) and histological analysis (3-week, 5-week and 7-week). Results indicated that there are differences occur following penetration between the resistant and susceptible. Those differences provide the first clues elucidating resistance mechanism in hybrid poplar stems. The results from this thesis will be used to improve resistance to Septoria canker in breeding programs.USDA-NIFANorth Dakota State University. College of Agriculture, Food Sciences and Natural Resource

The infection biology of Sphaerulina musiva: clues to understanding a forest pathogen.

Trees in the genus Populus and their interspecific hybrids are used across North America for fiber production and as a potential source of biofuel. Plantations of these species are severely impacted by a fungal pathogen, Sphaerulina musiva, the cause of leaf spot and stem canker. An inoculation protocol that does not rely on stem wounding to achieve infection was recently developed. Using this protocol two experiments were conducted to examine infection biology and disease development in the S. musiva-Populus interaction. In the first experiment non-wounded stems of one moderately resistant clone (NM6) and one susceptible clone (NC11505) were inoculated and examined by scanning electron microscopy at six different times (6 h, 12 h, 24 h, 72 h, 1 week, and 3 weeks) post-inoculation. The images indicate that the pathogen appears to enter host tissue through small openings and lenticels and that there are no significant differences in the penetration rate between the moderately resistant (NM6) and susceptible (NC11505) clones at 12 h post-inoculation. In a second experiment a histological comparison of stem cankers for resistant clone DN74 and susceptible clone NC11505 were conducted at three time points (3 weeks, 5 weeks, and 7 weeks) post-inoculation. Distinct differences in disease development were apparent between the resistant and susceptible clones at each time point, with the susceptible clone exhibiting a weak and delayed defense response. These results suggest, that following penetration, the pathogen may be able to interfere with the defense response in the susceptible host

Bright field micrographs of transverse sections through 7-week-old mock-inoculated controls of the resistant (DN74) and susceptible (NC11505) clones depicting the gross anatomy of stem tissue.

<p>(A) Stem anatomy of the mock-inoculated clone DN74. (B) Stem anatomy of mock inoculated clone NC11505. COX = Cortex, L = Lenticel, P = Periderm, PF = Phloem fiber. Scale bars = 200 µm.</p

Scanning electron micrographs (SEM) of the surface of mock-inoculated control trees at two different heights post-inoculation (PI).

<p>(A) Surface of upper 15 cm stem segment, lacking any openings/lenticels, collected from the susceptible clone NC11505. (B) Surface of upper 15 cm stem segment, lacking any openings/lenticels, collected from the moderately resistant clone NM6. (C) Surface of lower 15 cm stem segment, with small openings/lenticels indicated by the arrow, of clone NC11505. (D) Surface of lower 15 cm stem segment, with small openings/lenticels indicated by an arrow, of clone NM6. Magnification and scale bars included on the bottom of each image.</p

Scanning electron micrographs (SEM) of stems inoculated with a conidial suspension of <i>Sphaerulina musiva</i> at two different times (6 h and 12 h) post-inoculation (PI) depicting germination and penetration on the moderately resistant (NM6) and susceptible (NC11505) clones.

<p>(A) SEM micrograph of stem surface of NC11505 depicting <i>S. musiva</i> conidium 6 h PI. (B) SEM micrograph of stem surface of NM6 and conidium of <i>S. musiva</i> 6 h PI. (C) SEM micrograph of stem surface of NC11505 with conidium and germ tube of <i>S. musiva</i> entering a lenticel 12 h PI. (D) SEM micrograph of stem surface of NM6 with conidium and germ tube of <i>S. musiva</i> penetrating a small opening 12 h PI. (E) Micrograph (C) at increased magnification depicting penetration of lenticel by a germ tube. (F) SEM micrograph of stem surface of resistant clone NM6 with germ tube of <i>S. musiva</i> penetrating a small opening 12 h PI. Tr = trichome, Sp = conidium, L = lenticel, GT = germ tube, Op = small opening. Magnification and scale bars included on the bottom of each image.</p

A comparison of the mean, range, and standard deviation of penetration rates on the moderately resistant clone NM6 (<i>Populus maximowiczii</i>×<i>P. nigra</i>) and susceptible clone NC11505 (<i>P. maximowiczii</i>×<i>P. trichocarpa</i>) inoculated with a spore suspension of <i>Sphaerulina musiva</i>.

<p>The percentage of germ tubes entering natural openings was estimated by counting a total of 100 spores in two to three fields of view for each tree using a scanning electron microscope. Means were compared using a two-sample t-test (degrees of freedom = 1) means with the same letter are not significantly different (<i>P</i> = 0.41).</p

Bright field micrographs of susceptible clone (NC11505), at three different time points (3 weeks, 5 weeks, and 7 weeks) post-inoculation (PI) with blue stained hyphae, indicated by arrows, visible in different tissues.

<p>(A) Longitudinal section depicting hyphal growth in cortex at 3 weeks PI. (B) Transverse section through cortex depicting hyphal growth at 5 weeks PI. (C) Transverse section through xylem at 7 weeks PI depicting hyphal growth in xylem vessels (XV) and rays (XR). (D) Longitudinal section through xylem tissue depicting hyphal growth in vessels and rays. COX = Cortex, Pf = Primary phloem fiber, Pyc = Pycnidium, P = Periderm. Scale bars = 200 µm.</p

Transverse sections of susceptible clone NC11505 at 3 time points (3 weeks, 5 weeks, and 7 weeks) post-inoculation (PI) depicting anatomical responses to inoculation with a conidial suspension of <i>Sphaerulina musiva</i>.

<p>(A) Bright field micrograph of necrotic stem lesion 3 weeks PI. Necrotic area (Nec) bounded by layer of impervious tissue (IT indicated with arrow). (B) Fluorescent micrograph of necrotic lesion 3 weeks PI. IT layer visible as light purple fluorescence (indicated with arrows). (C) Bright field micrograph of necrotic lesion 5 weeks PI. Nec is bounded by a layer of necrophylactic periderm (NP; indicated with arrow). (D) Fluorescent micrograph of necrotic lesion 5 weeks PI. Nec is bounded by IT layer and NP layer (indicated with arrows). (E) Bright field micrograph of necrotic stem 7 weeks PI. Entire cortex (COX) is necrotic and filled with collapsed cells. (F) Fluorescent micrograph of necrotic lesion 7 weeks PI. NP and IT are absent from the Nec. Blue auto-fluorescence viewed under ultraviolet light. Filter parameters: Excitation filter G 365, Beam Splitter FT 395, Emission filter BP 445/50. Green auto-fluorescence viewed under ultraviolet light. Filter parameters: Excitation filter BP 450–490, Beam Splitter FT 510, Emission filter BP 515–565. P = periderm. Pf = primary phloem fiber. X = Xylem. VC = Vascular cambium. Scale bars = 200 µm.</p

Transverse sections of resistant clone DN74 at 3 time points (3 weeks, 5 weeks, and 7 weeks) post-inoculation (PI) depicting anatomical responses to inoculation with a conidial suspension of <i>Sphaerulina musiva</i>.

<p>(A) Bright field micrograph of necrotic lenticel (L) 3 weeks PI. Necrophylactic periderm (NP; indicated by an arrow) is apparent immediately below the infected L at the margin between the cortex (COX) and Periderm (P). (B) Fluorescent micrograph of necrotic lenticel 3 weeks PI. NP layer is evident as thick square shaped cells fluorescing bright purple immediately below the lenticel within the COX (indicated by an arrow). (C) Bright field micrograph of necrotic lesion, bounded by NP, 5 weeks PI. (D) Fluorescent micrograph of two necrotic lesions 5 weeks PI. NP is evident as thick layer of purple fluorescing cells (indicated by arrows) immediately below the necrotic area (Nec). (E) Bright field micrograph of Nec 7 weeks PI. Nec is bounded by two successive layers of NP (indicated by arrows), with occluded xylem (X) cells (dark red to black cells in the X) adjacent to the Nec. (F) Fluorescent micrograph of necrotic lesion with two successive layers of NP appearing purple (indicated by arrows). Blue auto-fluorescence viewed under ultraviolet light. Filter parameters: Excitation filter G 365, Beam Splitter FT 395, Emission filter BP 445/50. Green auto-fluorescence viewed under ultraviolet light. Filter parameters: Excitation filter BP 450–490, Beam Splitter FT 510, Emission filter BP 515–565. P = periderm. Pf = Primary phloem fiber. VC = Vascular Cambium. Scale bars = 200 µm.</p